JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2007, 58, 4, 845–857
www.jpp.krakow.pl
G. SIAWRYS, T. KAMINSKI, N. SMOLINSKA, J. PRZALA
EXPRESSION OF LEPTIN AND LONG FORM OF LEPTIN RECEPTOR
GENES AND PROTEINS IN PITUITARY OF CYCLIC
AND PREGNANT PIGS
Department of Animal Physiology, Faculty of Biology,
University of Warmia and Mazury in Olsztyn, Poland
Leptin is a multifunctional regulator in numerous tissues, including the pituitary. It is not
known, whether the porcine pituitary is a source of leptin synthesis and possesses the
leptin receptor protein. It is also unknown, if a relationship exists between expression
levels of these proteins in the pituitary and physiological status of sows. Therefore, the
aim of the study was 1] to examine, by Western-blotting analysis, the expression levels
of leptin and the long form of leptin receptor (OB-Rb) in the porcine anterior (AP) and
posterior (NP) pituitary gland during mid- and late-luteal phases of the oestrous cycle
(days 10 - 12 and 14 - 16) as well as during two stages of early pregnancy (days 14 - 16
and 30 - 32); and 2] to localise, using in situ hybridisation method (ISH), the expression
of leptin and OB-Rb genes in the pituitary gland in the above mentioned stages of the
cycle and pregnancy. Western-blotting analysis showed that leptin protein expression in
AP was higher in the late-luteal phase than in the mid-luteal phase, while OB-Rb protein
expression in both lobes was higher in the mid-luteal phase. In turn, during pregnancy
leptin protein content in AP and OB-Rb protein content in NP were more pronounced on
days 14 - 16 than on days 30 - 32. Comparison of leptin and OB-Rb protein expression
levels in AP between the mid-luteal phase and two periods of pregnancy showed,
respectively, stimulation of leptin protein and inhibition of OB-Rb protein expressions
during both examined stages of pregnancy. Taking AP from late-luteal phase as the point
of reference, it was revealed stimulation of leptin expression during earlier period of
pregnancy, whereas on days 30 - 32 of pregnancy both the hormone and its receptor
expressions
were
diminished.
In
turn,
comparison
of
leptin
and
OB-Rb
protein
expression levels in NP between the late-luteal phase and days 14 - 16 or 30 - 32 of
pregnancy showed inhibition of leptin protein expression and stimulation of OB-Rb
protein expression during pregnancy. Moreover, ISH studies localised leptin and OB-Rb
mRNAs expression in the cells of AP as well as NP tissue during the two stages of the
cycle and pregnancy. In conclusion, our findings suggest that leptin is produced within
the pituitary in the pig and may participate in auto/paracrine manner in the regulation of
this gland function during the luteal phase of the oestrous cycle and early pregnancy.
Key
words:
leptin, leptin receptor, pituitary, pig
846
INTRODUCTION
Leptin, discovered in 1994 as the ob gene product, is a protein with a molecular
mass of 16 kDa secreted mainly by adipose cells (1). It plays a key role in the
regulation of food intake and energy expenditure. Further studies revealed that leptin
is also a multifunctional paracrine regulator in numerous tissues, including pituitary
(2). Moreover, leptin is directly linked to reproductive processes through its actions
at various levels of the hypothalamic-pituitary-gonadal axis (HPG) (3 - 5). In target
tissues leptin binds to specific membrane receptors belonging to class I of the
cytokine receptor family (6). One of six isoforms of leptin receptor, long form (OBRb) is thought to be capable of full signal transduction and acts via the JAK (Janus
kinase)-STAT pathway (7). The expression of the OB-Rb gene has been detected
within the anterior pituitary (AP) in a number of species, including rats (8), mice (9),
sheep (10), pigs (11), men (12) which indicates the sensitivity of this gland to leptin.
Siawrys et al. (11) localised OB-Rb mRNA also in the porcine posterior pituitary
(NP). In turn, Morash et al. (13) detected leptin receptor immunoreactivity in the
anterior and intermediate lobes, but not in the posterior lobe of the neonatal rat
pituitary gland. Studies concerning leptin and OB-Rb gene expression in the porcine
pituitary were performed in prepurbertal, cyclic, pregnant animals and foetuses (11,
14, 15). Our previous experiments, using semiquantitative RT-PCR method (15),
showed different patterns of leptin and OB-Rb mRNAs expression in both lobes of
the porcine pituitary during the oestrous cycle and early pregnancy as well as the
increase of the OB-Rb gene expression in the pituitary during pregnancy compared
to the mid-luteal phase of the cycle. Moreover, in recent studies, using a quantitative
real time-PCR method we also indicated the expression of the gene for the short form
of leptin receptor (OB-Rs) in the pituitary (AP and NP) of cyclic and pregnant pigs
(16). OB-Rs gene expression level in the posterior pituitary, like OB-Rb transcript
content, was higher during pregnancy than the luteal phase of the cycle. However,
leptin and leptin receptor protein expressions have not been examined in the pituitary
of this species. Thus, it is still unclear, whether the porcine pituitary is sensitive to
leptin (possesses leptin receptor protein) and able to produce this peptide. Therefore,
the
aims
of
the
studies
were:
1]
to
determine,
by
Western-blotting
analysis,
expression levels of leptin and long form leptin receptor proteins in the porcine
pituitary gland (anterior - AP and posterior - NP lobes) during mid- and late-luteal
phases of the oestrous cycle (days 10 - 12 and 14 - 16) as well as during two early
stages of pregnancy (days 14 - 16 and 30 - 32) and 2] to confirm, using in situ
hybridisation method, the expression of leptin and OB-Rb genes in the pituitary
gland in the above mentioned stages of the cycle and pregnancy.
MATERIALS AND METHODS
Experimental animals
The studies were carried out in accordance with the principles and procedures of the Animal
Ethics Committee at the University of Warmia and Mazury in Olsztyn, Poland. Mature gilts (Large
847
White x Polish Landrace), at 9 - 10 months age, weighing 90 - 110 kg, descended from private
breeding were used. The animals were assigned to one of four experimental groups (n = 4 per
group) as follows: 1 and 2 - gilts killed on days 10 - 12 and 14 - 16 of the oestrous cycle, 3 and 4
- gilts inseminated on the first or second day of oestrus and killed on days 14 - 16 and 30 - 32 of
gestation. Pregnancy was confirmed by the presence of embryos/foetuses within uterine horns.
Within 10 min after slaughter, the adipose tissue, mediobasal hypothalamus (MBH) and the
pituitary gland were excised. The pituitary was separated into anterior and posterior parts. All
tissue samples were frozen in liquid nitrogen and maintained at -80°C until Western-blotting and
in situ hybridisation analysis.
Western-blotting analysis
The analysis was performed as described in a study by Smolinska et al. (17). The lobes of
pituitary were homogenised in a buffer (pH 7.6) containing: 50 mM NaCl, 10 mM Tris-HCl, 5 mM
EDTA, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 100 µM sodium orthovanadate,
0.02% sodium azide, 1% Triton X-100, 1 mM phenyl-methylsulphonyl fluoride and protease
inhibitors:
aprotinin
(2
µg/ml),
leupeptin
(2
µg/ml)
and
pepstatin
(1
µg/ml).
Subsequently,
homogenised tissues were incubated on ice for 30 min. After twice centrifugation at 10 000 g at 4
°C for 10 min, the supernatant containing the proteins was stored at -80 °C until required for use.
Then 100 µg (for OB-Rb and actin) or 150 µg (for leptin) of supernatant proteins were resolved by
SDS-PAGE (15% for leptin, 5% for OB-Rb and 12.5% for actin) and transferred to nitrocellulose
membranes (0.45 µm). After blocking in Tris-buffered saline with Tween-20 (TBST buffer: 10 mM
Tris, 150 mM NaCl, 0.05% Tween) containing 5% fat skimmed milk at 4 °C for 5 h, the membranes
were incubated with either rabbit polyclonal anti-leptin antibody (1:500; PA1
−
BioReagents, USA) or goat polyclonal anti-leptin receptor antibody (1:100; C-20 sc
052; Affinity
− 1832; Santa
Cruz Biotechnology, USA) or rabbit polyclonal anti-actin antibody (1:200; A 2066; Sigma, USA),
diluted in TBST buffer, at 4 °C overnight. The membranes were then washed with TBST buffer and
incubated with secondary alkaline phosphatase-conjugated antibodies (goat anti-rabbit for leptin
and actin or rabbit anti-goat for OB-Rb; AP sc
− 2057 and AP sc – 2949, respectively; Santa Cruz
Biotechnology, USA) diluted to 1:5000 in TBST buffer at room temperature (RT) for 1.5 h.
Nonspecific fetal calf serum (MP Biomedicals, USA) was used instead of primary antibodies to
produce negative control blots. After incubation and washing with TBST buffer, immunoreactive
bands were visualised using 4-nitroblue tetrazolium chloride (NBT, Promega, USA) and 5-bromo4-chloro-3-indolyl phosphate (BCIP, Promega, USA). Leptin/leptin receptor was quantified by
scanning densitometry of immunoblots. As a positive control for leptin and OB-Rb protein analysis
adipose tissue/mouse leptin and mediobasal hypothalamus (MBH) were used, respectively. The
choice of MBH was based on studies indicating the presence of OB-Rb in pig MBH (11, 18).
Statistics
Product yield was determined using GelScan for Windows ver. 1.45 (Kucharczyk, Poland).
Data were expressed as a ratio of leptin or OB-Rb protein content relative to actin protein in
arbitrary units. Respective mean values were compared within studied periods as well as between
the mid- or late-luteal phase and early pregnancy (days 14 - 16 – the beginning of implantation; or
days 30 - 32 – the end of implantation). All data were analysed by one-way ANOVA and least
significant
difference
(LSD)
post
hoc
test
and
are
reported
as
the
mean
±
SEM
from
four
independent observations. Statistical analyses were performed using Statistica program (Stat Soft
Inc., USA). Values for P
≤ 0.05 were considered statistically significant.
848
In situ hybridisation analysis
The protocol used for ISH for leptin and leptin receptor mRNA was described in our previous
study (17). Briefly, frozen sections (~ 6 µm) of porcine pituitary tissues (AP, NP) were attached to
poly-L-lysine-coated slides (Menzel-Glaser, Germany) and kept at - 80°C until required for use. Then
the sections were fixed with 4% paraformaldehyde in PBS, acetylated in 0.25% acetic anhydride in
0.1 M triethanolamine/0.9% NaCl, and dehydrated in graded ethanol (70%, 80%, 95%, and 100%).
After air-drying at RT, a hybridisation solution mixture (50% formamide, 4xSSC, 10% dextran sulfate,
1xDenhardt’s
antisense
solution,
0.5
mg/ml
salmon
S-labeled probe (~ 1.0 x 10
35
sperm
DNA,
0.25
mg/ml
tRNA)
which
contains
an
cpm/slide; leptin starter sequence: 5’ - TAG AGG GAG GCT
6
TCC AGG AC -3’; OB-Rb starter sequence: 5’ - TTG GGA TGC TGA TCT GAT AA - 3’) was
spotted on each slide. The slides were hybridised at 42°C for 22 h. The next day, the coverslips were
removed, and the slides were washed in 1xSSC at RT, followed by four washes in 2xSSC/50%
formamide at 40 - 45 °C and one washes in 1xSSC at RT. Next, the slides were dehydrated in graded
ethanol and air-dried at RT. All slides were coated with emulsion (LM-1, Amersham Bioscience, UK),
dried at RT and exposed at 4 °C for 21 days for leptin gene or 9 days for OB-Rb gene. Then samples
were developed (Eastman Kodak, USA), and counterstained with hematoxylin/eosin. As negative
controls sense probes were used. Localisation of mRNAs for leptin and its receptor was observed in
the optic microscope (CH30/CH40, Olympus, Japan) and archived by digital camera (C-5060 WZ,
Olympus, Japan) and program analySIS 5 Soft Imaging System (Olympus, Japan).
RESULTS
Western-blotting analysis of leptin protein expression
≤0.001) on days
In the AP, the leptin protein expression increased by 284% (p
14 - 16 of the cycle in comparison to expression of that protein on days 10 - 12
(Fig. 1A). Moreover, on days 10 - 12 and 14 - 16 of the cycle the content of leptin
≤0.001)
protein in NP was higher than in AP by 984% (p
respectively (data not presented).
≤0.001),
and 255% (p
On days 30 - 32 of pregnancy, leptin protein expression in AP was reduced by
≤0.001) compared to days 14-16 (Fig. 1A). Moreover, leptin expression in
≤0.001) than in
67% (p
NP during the later period of pregnancy was higher by 279% (p
AP (data not shown).
Both on days 14 - 16 and 30 - 32 of early pregnancy leptin protein expression
in the AP was significantly higher than on days 10 - 12 of the cycle (by 664%;
p
≤0.001
and
pregnancy
by
151%;
expression
of
≤0.01,
p
leptin
respectively).
protein
in
AP
Moreover,
was
higher
on
by
days
99%
14-16
of
≤0.01)
(p
compared to days 14 - 16 of the cycle, whereas the expression on days 30-32 of
≤0.01) than on days 14 - 16 of the cycle (Fig. 1A).
pregnancy was lower by 35% (p
Furthermore, leptin protein expression in the NP was reduced on days 14-16 and
≤0.05
30 - 32 of early pregnancy relative to days 14 - 16 of the cycle (by 32%; p
≤0.01, respectively) (Fig. 1B).
and by 30%; p
Western-blotting analysis of OB-Rb protein expression
During the late-luteal phase of the cycle (days 14 - 16), expression of the leptin
≤0.001) and 88%
receptor in AP (Fig. 2A) and NP (Fig. 2B) decreased by 40% (p
849
A
A
MM
AP
AP
AP
AP
10-12
14-16
14-16
30-32
ADIPOSE
TISSUE
MOUSE
LEPTIN
ACTIN (42 kDa)
LEPTIN
(16 kDa)
DAYS 14-16 OF THE
OESTROUS CYCLE
DAYS 10-12 OF THE
OESTROUS CYCLE
DAYS 14-16 OF
PREGNANCY
DAYS 30-32 OF
PREGNANCY
B
B
MM
NP
10-12
NP
NP
14-16
14-16
NP
30-32
ADIPOSE
MOUSE
TISSUE
LEPTIN
ACTIN (42 kDa)
LEPTIN
(16 kDa)
DAYS 10-12 OF THE
OESTROUS CYCLE
DAYS 14-16 OF THE
OESTROUS CYCLE
DAYS 14-16 OF
PREGNANCY
DAYS 30-32 OF
PREGNANCY
Fig. 1. Western-blotting analysis of leptin protein expression in the pituitary [A - anterior (AP) and
B - posterior (NP) parts] of cyclic (days 10 - 12 and 14 - 16) and pregnant (days 14 - 16 and 30 32) pigs. Upper panels: transfer of leptin protein and actin protein on the nitrocellulose membrane
(MM – molecular marker, adipose tissue and mouse leptin as a positive control); lower panels:
densitometric analysis of leptin protein relative to actin protein. Data are means (± SEM) for four
animals. Differences in protein levels were assumed as statistically significant for P<0.05 and
marked with different letters.
850
≤0.05),
(p
respectively, compared to the mid-luteal phase. Furthermore, on days
14 - 16 of the cycle the content of OB-Rb protein within AP was higher by 670%
≤0.001) than in NP (data not presented).
(p
On days 30 - 32 of pregnancy, leptin receptor expression in NP was diminished
≤0.05) compared to the respective protein expression observed on days
by 57% (p
14 - 16 (Fig. 2B). Furthermore, at the later stage of pregnancy OB-Rb protein
≤0.001) than in NP (data not shown).
content in AP was higher by 103% (p
In contrast to leptin protein, OB-Rb protein expression detected in AP on days
≤0.05) and 49% (p≤0.001),
14 - 16 and 30 - 32 of pregnancy was lower by 30% (p
respectively, than the expression in mid-luteal phase of the cycle. Moreover, the
receptor expression in AP on days 30 - 32 of pregnancy was also reduced (by
≤0.01)
16%, p
compared to days 14 - 16 of the cycle (Fig. 2A). In turn, OB-Rb
protein expression detected in NP on days 14 - 16 and 30-32 of pregnancy was
≤0.01) and 220% (p≤0.001), respectively, than the expression
higher by 650% (p
on days 14 - 16 of the cycle (Fig. 2B).
ISH localisation of leptin and leptin receptor mRNAs in porcine pituitary
ISH studies localised leptin and OB-Rb mRNAs in the cells of both pituitary
lobes during the mid-luteal (days 10 - 12) and late-luteal (days 14 - 16) phases of
the cycle as well as during the two stages of early pregnancy (days 14 - 16 and
30-32) (Fig. 3 and 4, respectively). Hybridisation was also found in adipose tissue
used as a positive control for leptin and in MBH applied as a positive control for
the leptin receptor. When the sense probe was used, no silver grains were detected
in either the pituitary or control tissues.
DISCUSSION
The present study, using Western-blotting analysis, reveals for the first time
that leptin and its receptor proteins are expressed in the pituitary gland (AP and
NP) of the pig. Moreover, the results of this study demonstrate changes in leptin
protein and leptin receptor protein expression in the porcine pituitary during the
oestrous cycle and early pregnancy. The obtained results indicate that during early
pregnancy, stimulation of leptin protein expression takes place in the AP, which
confirm our earlier studies (15) suggesting leptin and OB-Rb gene expression in
pituitaries of cyclic and pregnant pigs.
The detection of leptin protein expression within the pituitary during the cycle
and pregnancy suggests that this gland, besides adipose tissue, is also one of
leptin source. The localisation of leptin and its receptor mRNAs expression in the
porcine pituitary, using hybridisation in situ method, confirms this hypothesis and
supports earlier studies indicating both genes’ expression in porcine (15), rodent
(9, 13, 19, 20), sheep (10) and human (12) pituitaries.
851
A
A
MM
AP
10-12
AP
AP
AP
14-16
14-16
30-32
MBH
ACTIN (42 kDa)
OB-Rb
(170 kDa)
DAYS 10-12 OF THE
OESTROUS CYCLE
B
DAYS 14-16 OF
PREGNANCY
DAYS 30-32 OF
PREGNANCY
B
DAYS 14-16 OF THE
OESTROUS CYCLE
MM
NP
NP
NP
NP
10-12
14-16
14-16
30-32
MBH
ACTIN (42 kDa)
OB-Rb
(170 kDa)
DAYS 10-12 OF THE
OESTROUS CYCLE
DAYS 14-16 OF THE
OESTROUS CYCLE
DAYS 14-16 OF
PREGNANCY
DAYS 30-32 OF
PREGNANCY
Fig. 2. Western-blotting analysis of leptin receptor protein expression in the pituitary [A - anterior
(AP) and B - posterior (NP) parts] of cyclic (days 10 - 12 and 14 - 16) and pregnant (days 14 - 16
and 30 - 32) pigs. Upper panels: transfer of leptin receptor protein and actin protein on the
nitrocellulose membrane (MM – molecular marker, MBH as a positive control); lower panels:
densitometric analysis of leptin receptor protein relative to actin protein. Data are means (± SEM)
for four animals. Differences in protein levels were assumed as statistically significant for P<0.05
and marked with different letters.
852
Fig. 3. In situ hybridisation analysis (dark-field images) of leptin mRNA in the pituitary (AP anterior and NP - posterior parts) of cyclic (days 10 - 12 and 14 - 16) and pregnant (days 14 - 16
and 30 - 32) pigs. A bright-field photomicrographs depict histological preparation of tissues stained
by hematoxylin/eosin. Insets – corresponding tissues hybrised with radiolabeled sense probe (sense)
served as a negative control. A positive control of leptin gene expression was adipose tissue (AT).
Magnification 250x.
Interestingly, the expression pattern of leptin and OB-Rb proteins (present
study) and expressions of corresponding genes (15) in the porcine pituitary during
the same periods of the oestrous cycle and pregnancy are often quit different. For
example, in contrast to leptin transcript level, the protein content in the AP
increased during the later stage of pregnancy (days 30 - 32) compared to the midluteal phase of the cycle (days 10 - 12). In turn, unlike OB-Rb mRNA expression,
the OB-Rb protein expression in AP was reduced on days 14 - 16 of pregnancy
compared
to
days
posttranscriptional
10
-
12
of
processing
the
of
cycle.
leptin
This
and
its
suggests
the
receptor
genes’
existence
of
products
the
and
presence of the intracellular regulatory mechanism of the genes’ expression. An
inverse relationship was also observed for the levels of leptin and its receptor in
the pig pituitary, which implies homologous regulation of the leptin receptor by
leptin
itself.
Similar
homologous
regulation
of
the
leptin
receptor,
on
the
853
Fig. 4. In situ hybridisation analysis (dark-field images) of OB-Rb mRNA in the pituitary (AP anterior and NP - posterior parts) of cyclic (days 10 - 12 and 14 - 16) and pregnant (days 14 - 16
and 30 - 32) pigs. A bright-field photomicrographs depict histological preparation of tissues stained
by hematoxylin/eosin. Insets – corresponding tissues hybrised with radiolabeled sense probe (sense)
served
as
a
negative
control.
A
positive
control
of
OB-Rb
gene
expression
was
mediobasal
hypothalamus (MBH). Magnification 250x.
transcript level, was described for the porcine pituitary (15) and previously for the
rat hypothalamus (21) and testis (22). Furthermore, hypothalamic and peripheral
factors may also participate in regulating both OB-Rb and leptin expression. It
was shown that GHRH decreases long form leptin receptor expression in porcine
anterior pituitary cells (23). On the other hand, GHRH added with oestradiol
increased the percentage of pituitary cells with leptin protein or mRNA (24).
Recently Akhter et al. (25) revealed that GnRH and estrogen added together
increased the coexpression of leptin mRNA and gonadotrophins in rat anterior
pituitary. They also detected changes in the percentage of AP cells expressing
leptin protein relative to the phase of the cycle and period of pregnancy. It
indicates that the ability of pituitary gland to produce leptin is dependent on
hormonal status of animals. The same authors observed that AP leptin mRNA
expression increases from metestrous to diestrous followed by decrease on the
854
morning of proestrous. They also noticed that cellular leptin protein level in AP
was
increased
by
GnRH
treatment
in
proestrous
rats
and
GnRH
stimulated
secretion of leptin by the cells from diestrous, proestrous and pregnant rats. The
authors suggested that high level of AP leptin expression during proestrus and
pregnancy support its participation during key events involved with reproduction.
Results of our study also shown changes of leptin protein expression levels in the
porcine pituitary between the oestrous cycle and pregnancy (increase of leptin
protein expression level in AP during pregnancy) and confirm above hypothesis.
Leptin
is
produced
by
different
pituitary
cells.
Colocalisation
studies
in
humans with leptin and pituitary hormones revealed that 70% of ACTH cells,
21% of GH cells, 29% of LH cells, 33% of FSH cells, 32% of TSH cells, 64% of
folliculostellate (FS) cells and only very few PRL cells (3%) were positive for
leptin (12). In rats the major cell type colocalised with leptin was TSH cell (24%),
whereas a smaller percentage of LH (4%) and FSH (3%) cells were positive for
that protein. Moreover, less than 1% of ACTH, GH, PRL and FS cells expressed
leptin by colocalisation studies (9). Leptin production in the pituitary gland was
also noted in mice (9) and cattle (26). Cellular localisation of OB-R in the
pituitary
was
indicated
as
well;
however,
like
in
the
case
of
leptin,
species
differences in the localisation of this receptor were observed. Notably, in the rat
anterior
pituitary,
GH-secreting
cells
almost
exclusively
possessed
leptin
receptors, while leptin receptors in TSH, LH, and FSH cells were difficult to
localise (20). In the sheep, leptin receptors were found in GH (69%), ACTH
(27%) and LH (29%) cells of pars distalis of the anterior pituitary as well as in
LH cells (90%) of the pars tuberalis (27). Moreover, FS cells and various rat and
mouse pituitary cell lines have also been reported to express leptin receptors (9,
28). It is still unclear, what type of cells in the porcine pituitary produces leptin
and possesses OB-Rb. Results of the present study did not solve this problem.
The presence of the leptin receptor protein in the porcine pituitary might
suggest that leptin is involved in the regulation of its function and, in connection
with local leptin synthesis findings, further supports the hypothesis that leptin
acts as an autocrine/paracrine factor within this gland. It is worth noting however,
that also adipocyte-derived leptin may bind to the receptors and affect pituitary
functions. In anterior pituitary leptin is involved in regulation of growth and
differentiation of pituitary cells. High leptin concentration was reported to inhibit
cell
proliferation
in
human
and
rat
pituitary
cell
lines
(12).
In
vitro
studies
indicated that this hormone stimulated secretion of GH (29), LH, FSH, PRL (3032) but inhibited TSH secretion (33). The leptin effect on the release of pituitary
hormones may not only be direct, but also indirect. For example, it was shown
that nitric oxide, whose production is affected by leptin, might be involved in the
stimulation of LH, FSH and GH secretion (29, 32, 34). By hand of the pituitary
hormones, leptin can effect on function of peripheral endocrine organs, including
gonads, adrenals, thyroid and others. Moreover, it was found that leptin is costored
with
pituitary
hormones
in
the
same
secretory
granules
and
is
855
concomitantly released with them in a pulsatile manner (9, 35). Untill now, it has
not been known the possible role of leptin in posterior pituitary. Our studies
showed that leptin is produced in this lobe of the pituitary and the level of leptin
protein expression in the NP during the cycle and pregnancy is higher than in the
AP. It is conceivable, that this hormone participates in the control of posterior
pituitary function, among others in the regulation of oxytocin and/or vasopressin
release. However, this hypothesis remains to be verified.
In summary, the presented studies indicated for the first time that leptin and its
receptor proteins are expressed in the pituitary gland (AP and NP) in the pig and
revealed different patterns of these proteins’ expression during the luteal phase of
the cycle and early pregnancy. Moreover, the studies localised leptin and OB-Rb
mRNAs expression in the cells of AP as well as NP tissue both during examined
periods of the cycle and pregnancy. These findings suggest that leptin is produced
in the porcine pituitary and may participate in auto/paracrine regulation of pituitary
functions during the luteal phase of the oestrous cycle and early pregnancy.
Acknowledgement: This research was supported by the State Committee for Scientific Research
(projects: No PBZ KBN-084/P06/2002 and No 0206.0805).
REFERENCES
1.
Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the
mouse obese gene and its human homologue. Nature 1994; 372: 425-432.
2.
Fruhbeck G. A heliocentric view of leptin. Proc Nutr Soc 2001; 60: 301-318.
3.
Caprio
M,
Fabbrini
E,
Isidori
AM,
Aversa
A,
Fabbri
A.
Leptin
in
reproduction.
Trend
Endocrinol Metab 2001; 12: 65-72.
4.
Barb CR, Hausman GJ, Czaja K. Leptin: a metabolic signal affecting central regulation of
5.
Zieba
reproduction in the pig. Domest Anim Endocrinol 2005; 29:186-192.
DA,
Amstalden
M,
Williams
GL.
Regulatory
roles
of
leptin
in
reproduction
and
metabolism: a comparative review. Domest Anim Endocrinol 2005; 29: 166-185.
6.
Tartaglia LA, Demski M, Weng X, et al. Identification and expression cloning of a leptin
7.
Hakansson ML, Meister B. Transcription factor STAT3 in leptin target neurons of the rat
8.
Zamorano PL, Mahesh VB, De Sevilla LM, Chorich LP, Bhat GK, Brann DW. Expression and
receptor, OB-R. Cell 1995; 83: 1263-1271.
hypothalamus. Neuroendocrinology 1998; 68: 420-427.
localization
of
the
leptin
receptor
in
endocrine
and
neuroendocrine
tissues
of
the
rat.
Neuroendocrinology 1997; 65: 223-228.
9.
Jin L, Zhang S, Burguera BG, et al. Leptin and leptin receptor expression in rat and mouse
pituitary cells. Endocrinology 2000; 141: 333-339.
10. Dyer CJ, Simmons JM, Matteri RL, Keisler DH. Leptin receptor mRNA is expressed in ewe
anterior pituitary and adipose tissues and is diferentially expressed in hypothalamic regions of
well-fed and feed-restricted ewes. Domest Anim Endocrinol 1997; 14: 119-128.
11. Siawrys G, Przala J, Kaminski T, et al. Long form leptin receptor mRNA expression in the
hypothalamus and pituitary during early pregnancy in the pig. Neuro Endocrinol Lett 2005; 26:
305-309.
856
12. Jin L, Burguera BG, Couce ME, et al. Leptin and leptin receptor expression in normal and
neoplastic
human
pituitary.
Evidence
of
a
regulatory
role
for
leptin
on
pituitary
cell
proliferation. J Clin Endocrinol Metab 1999; 84: 2903-2911.
13. Morash B, Imran A, Wilkinson D, Ur E, Wilkinson M. Leptin receptors are developmentally
regulated in rat pituitary and hypothalamus. Mol Cell Endocrinol 2003; 210: 1-8.
14. Lin J, Barb CR, Matteri RL, et al. Long form leptin receptor mRNA expression in the brain,
pituitary and other tissues in the pig. Domest Anim Endocrinol 2000; 19: 53-61.
15. Kaminski T, Smolinska N, Gajewska A, et al. Leptin and long form of leptin receptor genes
expression in the hypothalamus and pituitary during the luteal phase and early pregnancy in
pigs. J Physiol Pharmacol 2006; 27: 95-108.
16. Bogacka I, Przala J, Siawrys G, Kaminski T, Smolinska N. The expression of short from of
leptin receptor gene during early pregnancy in the pig examined by quantitative real time RTPCR. J Physiol Pharmacol 2006; 57: 479-489.
17. Smolinska N, Kaminski T, Siawrys G, Przala J. Long form of leptin receptor gene and protein
expression in the porcine ovary during the estrous cycle and early pregnancy. Reprod Biol 2007;
7: 17-39.
18. Lin J, Barb R, Kraeling RK, Rampacek GB. Developmental changes in the long form leptin
receptor and related neuropeptide gene expression in the pig brain. Biol Reprod 2001; 64:
1614-1618.
19. Morash B, Li SA, Murphy PR, Wilkinson M, Ur E. Leptin gene expression in the brain and
pituitary gland. Endocrinology 1999; 140: 5995-5998.
20. Sone M, Nagata H, Takekoshi S, Osamura RY. Expression and localization of leptin receptor in
normal rat pituitary gland. Cell Tissue Res 2001; 305: 351-356.
21. del Carmen Garcia M, Casanueva FF, Dieguez C, Senaris RM. Gestational profile of leptin
messenger ribonucleic acid (mRNA) content in the placenta and adipose tissue in the rat, and
regulation
of
mRNA
levels
of
the
leptin
receptor
subtypes
in
the
hypothalamus
during
pregnancy and lactation. Biol Reprod 2000; 62: 698-703.
22. Tena-Sempere M, Pinilla L, Zhang FP, et al. Developmental and hormonal regulation of
leptin receptor (Ob-R) messenger ribonucleic acid expression in rat testis. J Endocrinol
2001; 170: 413-423.
23. Lin J, Barb CR, Kraeling RR, Rampacek GB. Growth hormone releasing factor decreases long
form leptin receptor expression in porcine anterior pituitary cells. Domest Anim Endocrinol
2003; 24: 95-101.
24. McDuffie IA, Akhter N, Childs GV. Regulation of leptin mRNA and protein expression in
pituitary somatotropes. J Histochem Cytochem 2004; 52: 263-273.
25. Akhter N, Johnson BW, Crane C, et al. Anterior pituitary leptin expression changes in different
reproductive
states:
in
vitro
stimulation
by
gonadotropin-releasing
hormone.
J
Histochem
Cytochem 2007; 55: 151-166.
26. Yonekura S, Senoo T, Kobayashi Y, Yonezawa T, Katoh K, Obara Y. Effects of acetate and
butyrate on the expression of leptin and short-form leptin receptor in bovine and rat anterior
pituitary cells. Gen Comp Endocrinol 2003; 133: 165-172.
27. Iqbal
J,
Pompolo
S,
Considine
RV,
Clarke
IJ.
Localization
of
leptin
receptor-like
immunoreactivity in the corticotropes, somatotropes, and gonadotropes in the ovine anterior
pituitary. Endocrinology 2000; 141: 1515-1520.
28. Jin L, Tsumanuma I, Ruebel KH, Bayliss JM, Lloyd RV. Analysis of homogeneous populations
of
anterior
pituitary
folliculostellate
cells
by
laser
capture
microdissection
transcription-polymerase chain reaction. Endocrinology 2001; 142: 1703-1709.
and
reverse
857
29. Baratta M, Saleri R, Mainardi GL, Valle D, Giustina A, Tamanini C. Leptin regulates GH gene
expression and secretion and nitric oxide production in pig pituitary cells. Endocrinology 2002;
143: 551-557.
30. Barb CR, Barrett JB, Kraeling RR. Role of leptin in modulating the hypothalamic-pituitary axis and
luteinizing hormone secretion in the prepuberal gilt. Domest Anim Endocrinol 2004; 26: 201-214.
31. Tezuka M, Irahara M, Ogura K, Kiyokawa M, Tamura T, Matsuzaki T. Effects of leptin on
gonadotropin secretion in juvenile female rat pituitary cells. European J Endocrinol 2002; 146:
261-266.
32. McCann SM, Kimura M, Walczewska A, Karanth S, Rettori V, Yu WH. Hypothalamic control
of
gonadotropin
secretion
by
LHRH,
FSHRF,
NO,
cytokines
and
leptin.
Domest
Anim
Endocrinol 1998; 15: 333-344.
33. Ortiga-Carvalho TM, Oliveira KJ, Soares BA, Pazos-Moura CC. The role of leptin in the regulation
of TSH secretion in the fed state: in vivo and in vitro studies. J Endocrinol 2002; 174: 121-125.
34. Kosior-Korzecka
U,
Bobowiec
R.
Leptin
effect
on
nitric
oxide
and
GnRH-induced
FSH
secretion from ovine pituitary cells in vitro. J Physiol Pharmacol 2006; 57: 637-639.
35. Bagnasco M, Kalra PS, Kalra SP. Plasma leptin levels are pulsatile in adult rats: effects of
gonadectomy. Neuroendocrinology 2002; 75: 257-263.
Received:
August 2, 2007
A c c e p t e d : November 5, 2007
Author’s address: Phone: +48 89 5233201; fax:+48 89 5233937; e-mail: gabri@uwm.edu.pl
(Gabriela Siawrys)