JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2002, 53, 3, 439–451
www.jpp.krakow.pl
I. BOGACKA, G. SIAWRYS, S. OKRASA, T. KAMINSKI, J.PRZALA
THE INFLUENCES OF GNRH, OXYTOCIN AND VASOACTIVE
INTESTINAL PEPTIDE ON LH AND PRL SECRETION BY PORCINE
PITUITARY CELLS IN VITRO.
Department of Animal Physiology, University of Warmia and Mazury in Olsztyn, 10-718
Olsztyn-Kortowo 5, Poland.
The aim of the present study was to evaluate the possible direct effects of GnRH,
oxytocin (OT) and vasoactive intestinal peptide (VIP) on the release of LH and PRL
by dispersed porcine anterior pituitary cells. Pituitary glands were obtained from
mature gilts, which were ovariektomized (OVX) one month before slaughter. Gilts
randomly assigned to one of the four groups were treated: in Group 1 (n=8) with 1
ml/100 kg b.w. corn oil (placebo); in Group 2 (n=8) and Group 3 (n=8) with estradiol
benzoate (EB) at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h
before slaughter; and in Group 4 (n=9) with progesterone (P4) at the dose 120 mg/100
kg b.w. for five consecutive days before slaughter. In gilts of Group 2 and Group 3
treatments with EB have induced the negative and positive feedback in LH secretion,
respectively. Isolated anterior pituitary cells (106/well) were cultured in McCoy's 5a
medium with horse serum and fetal calf serum for 3 days at 37°C under the
atmosphere of 95% air and 5% CO2. Subsequently, the culture plates were rinsed
with fresh McCoy's 5A medium and the cells were incubated for 3.5 h at 37°C in the
same medium containing one of the following agents: GnRH (100 ng/ml), OT (101000 nM) or VIP (1-100 nM). The addition of GnRH to cultured pituitary cells
resulted in marked increases in LH release (p<0.001) in all experimental groups. In
the presence of OT and VIP we noted significant increases (p<0.001) in LH secretion
by pituitary cells derived from gilts representing the positive feedback phase (Group
3). In contrast, OT and VIP were without any effect on LH release in Group 1
(placebo) and Group 2 (the negative feedback). Pituitary cells obtained from OVX
gilts primed with P4 produced significantly higher amounts (p<0.001) of LH only
after an addition of 100 nM OT. Neuropeptide GnRH did not affect PRL secretion by
pituitary cells obtained from gilts of all experimental groups. Oxytocin also failed to
alter PRL secretion in Group 1 and Group 2. However, pituitary cells from animals
primed with EB 60-66 h before slaughter and P4 produced markedly increased
amounts of PRL in the presence of OT. Neuropeptide VIP stimulated PRL release
from pituitary cells of OVX gilts primed with EB (Groups 2 and 3) or P4. In contrast,
in OVX gilts primed with placebo, VIP was without any effect on PRL secretion. In
conclusion, the results of our in vitro studies confirmed the stimulatory effect of
440
GnRH on LH secretion by porcine pituitary cells and also suggest a participation of
OT and VIP in modulation of LH and PRL secretion at the pituitary level in a way
dependent on hormonal status of animals.
K e y w o r d s : GnRH, oxytocin, VIP, prolactin, LH, pigs, pituitary cells
INTRODUCTION
It has been widely documented that some neuropeptides play an important role
in the mechanism of hormone release from the pituitary gland. The secretion of
LH from adenohypophysis in many species is predominantly controlled by
stimulatory action of the hypothalamic neuropeptide, GnRH (1-3). It has been
well established that prolactin (PRL) secretion is restrained by PRL-inhibiting
factors (PIF), of which the most important is dopamine (4). Several evidences
suggest that PRL release may also be regulated by one or more hypothalamic
PRL-releasing factors (5, 6). The secretion of both PRL and LH is additionally
modulated by ovarian steroids, 17β-estradiol (E2) and progesterone (P4), acting
directly at the pituitary level as well as indirectly through the hypothalamus.
Estradiol has been reported to have a biphasic effect on LH secretion in pigs (7,
8). The administration of estradiol benzoate (EB) to OVX sows initially reduces
plasma LH concentrations (for approx. 50 h) and pituitary responsiveness to
GnRH. Thereafter, increasing frequency of GnRH pulses released from the
median eminence and elevated number of pituitary receptors for GnRH are
observed, and finally a preovulatory-like surge of LH is induced (7-9). Estrogens
are also believed to be important modulators of PRL secretion by the pituitary
gland. A preovulatory rise in plasma E2 concentration is obligatory for generating
the proestrous surge of PRL in rats (10). Estrogens exert a direct effect on the
pituitary gland by stimulating PRL gene transcription, PRL synthesis, storage,
and secretion. Estrogens also stimulate proliferation of PRL producing cells (10,
11). Less is known about effects of P4 on the regulation of LH and PRL secretion
in the pig. Data pertaining to this aspect of P4 action in other species are divergent.
Several studies demonstrated that P4 both stimulated and inhibited PRL and LH
secretion (12-14). Moreover, P4 can partly reverse the stimulatory effect of
estrogens on the pituitary hormone secretion (15, 16).
Extensive studies, primarily on ruminants and laboratory rodents, have
established that, besides gonadal steroids, oxytocin (OT) and vasoactive intestinal
peptide (VIP) are involved in the control of LH and PRL secretion. These studies
have confirmed: 1/ the OT and VIP ability to increase PRL (17-19) and LH
secretion (3, 20, 21); 2/ the presence of OT and VIP at high concentrations in the
hypophysial portal vasculature connecting the median eminence with the anterior
pituitary (22, 23); and 3/ the localisation of OT (OTR) and VIP (VIPR) receptors
441
in the anterior pituitary gland (24, 25). Moreover, a possibility of OT counter
current transfer from venous blood of the perihypophyseal cavernous sinus to the
arterial blood of the carotid rete, supplying the brain and hypophysis, was
demonstrated in cyclic pigs (26). It has also been shown that concentrations of OT
in portal blood vary throughout the estrous cycle in rats (27). The highest OT
levels were observed in rats on the day of proestrus (before ovulation) in
correlation with the preovulatory surges of LH and PRL. Nevertheless, an
engagement of these peptides in the regulation of LH and PRL release from
pituitary cells remains unclear in rats and unknown in pigs.
Therefore, the aim of the present study was to evaluate the possible effects of
GnRH, OT and VIP on the release of LH and PRL employing the anterior
pituitary cells isolated from OVX gilts treated in vivo with placebo, EB or P4.
MATERIALS AND METHODS
Chemicals
Dulbecco's medium, McCoy's-5a medium, gonadotropin releasing hormone (GnRH),
vasoactive intestinal peptide (VIP), nystatin, MEM-non-essential amino acids, bacitracin were
purchased from Sigma (St. Louis, MO, USA). Labelled hormones: (1,2,6,7-3H) progesterone and
(2,4,6,7-3H) estradiol were from Amersham, UK. Steroids (estradiol benzoate, progesterone) and
antibiotics (penicillin, streptomycin) were obtained from Polfa (Poland). Trypsin was a product of
the Laboratory of Sera and Vaccines (Lublin, Poland), BSA fraction V was from the Laboratory of
Sera and Vaccines (Cracow, Poland). Fetal calf serum was purchased from Plastomed (Poland) and
trypan blue from Chemapol (Czech Republic). Succinyl-oxytocin was kindly provided by Prof.
Zbigniew Grzonka (Institute of Chemistry, University of Gdansk, Poland) and PRL (30 IU/mg) was
isolated from porcine pituitaries and kindly provided by Prof. K. Kochman (Institute of Animal
Physiology and Nutrition, Polish Academy of Sciences, Jablonna, Poland). Other reagents not
mentioned in the text were from Sigma.
Animals and experimental procedures
The studies were carried out in accordance with the principles and procedures of the Animal
Ethics Committee at the University of Warmia and Mazury. Sexually mature crossbred gilts with an
average body weight (b.w.) of 90 kg (80-100) and 6-8 months of age originated from a commercial
farm. Pigs were ovariectomized under general anaesthesia 30 days before the beginning of
experiments. Gilts randomly assigned to one of the four groups were treated: in Group 1 (OVX;
n=8) with 1 ml/100 kg b.w. corn oil (placebo); in Group 2 (OVX+EB I; n=8) and Group 3
(OVX+EB II; n=8) with EB at the dose 2.5 mg/100 kg b.w., respectively, 30-36 h and 60-66 h
before slaughter; and in Group 4 (OVX+P4; n=9) with P4 at the dose 120 mg/100 kg b.w. for five
consecutive days before slaughter.
It is known that treatment of OVX gilts with EB results in biphasic response in LH secretion (8,
28). Initially, the negative feedback occurs, which is followed by the positive feedback phase,
comprising estradiol-induced LH surge indistinguishable from the preovulatory surge. In our
experiment gilts of Groups 2 and 3 represented periods of the negative and positive feedback,
respectively. Treatment of pigs with P4 (Group 4) mimicked luteal phase of the estrous cycle.
442
For confirmation of hormonal status of experimental gilts, blood samples were collected during
slaughter to measure LH, E2, and P4 plasma concentrations. The blood samples were placed in
chilled heparinized tubes and centrifuged at 2500×g. Plasma was harvested and stored at -20°C until
assayed for LH and steroid hormones by RIA.
Preparation of pituitary cells and in vitro experiments
The procedure of pituitary tissue dissociation, cell preparation and cell culture were based on
methods described previously (29). Briefly, the anterior lobes were dissected from each pituitary,
minced into small pieces (1-2 mm) and washed several times with Dulbecco's medium. Single cell
suspension of anterior pituitaries was then prepared by 0.3% trypsin digestion at 37°C for 8-10 min.
The dispersed pituitary cells transferred to a plastic tube were repeatedly centrifuged at 800×g for
8 min and washed with Dulbecco's medium (three times). After the final wash, the cell suspension
was passed through a nylon filter (60 µm mesh) to remove undigested fragments of the tissue and
the cells were counted in a hemocytometer. Cell viability (97-98%) was determined by trypan blue
dye exclusion. Finally, pituitary cells were resuspended in McCoy's 5A medium containing 10%
horse serum, 2.5% fetal calf serum, 240 U/ml nystatin, 100 U/ml penicillin, and 100 µg/ml
streptomycin. One ml suspension of dispersed cells (1×106/ml) was transferred to each culture dish
of 24-well plates and pre-incubated for 3 days at 37°C under a water-saturated atmosphere of 5%
CO2 and 95% air. The cells, which attached to the dishes, were then washed twice with 1 ml fresh
McCoy's 5A medium without serum. After the final wash, the cells were incubated (3.5 h at 37°C)
in 1 ml McCoy's medium with bacitracin (2×10-5 M), without (control culture) or with following
agents: GnRH (100 ng/ml), OT (10-1000 nM) or VIP (1-100 nM). All incubations were performed
in duplicates. At the end of the incubation, media were collected and stored at - 20°C until RIA
analyses.
Hormone assay
Radioimmunoassay of LH
LH concentrations in the culture media and plasma were established by the RIA procedure
previously described by Ziecik et al. (30). Rabbit antibodies against porcine LH were used and the
cross-reactions were published previously (31). The sensitivity of the assay and the intra- and interassay coefficients of variation were 0.08 ng/ml, 9.4% and 11.2%, respectively.
Radioimmunoassay of PRL
PRL concentration in the culture media was analysed according to the method described by
Dusza & Krzymowska (32). Goat antibodies against porcine PRL were used, which did not exhibit
cross-reactivity with porcine LH and FSH. The sensitivity of the assay was 0.09 ng/ml and the intraand inter-assay coefficients of variation were 4.4% and 6.6%, respectively.
Radioimmunoassays of steroid hormones
17β-estradiol plasma concentration was determined according to the method of Hotchkiss et al.
(33). Cross-reactivity of the antisera against E2 was previously published by Szafranska & Tilton
(31). The sensitivity of the assay and the intra-assay coefficient of variation were 10.3 pg/ml and
6.3%, respectively. The inter-assay coefficient of variation was not established, since the hormone
concentration was determined in one run.
443
Progesterone plasma concentration was determined by the procedure previously described by
Ottobre et al. (34). Cross-reactivity of the antisera against P4 was described by Dziadkowiec et al.
(35). The sensitivity of the assay and the intra-assay coefficient of variation were 22 pg/ml and
5.7%, respectively. The P4 concentration was determined in one run and the inter-assay coefficient
of variation for this hormone was not established.
Statystical analysis
Data from in vivo experiments are expressed as means ±SEM. Since high differences in the
amounts of LH and PRL secreted in vitro by pituitary cells of different gilts had been noted, the
data were expressed as a percentage (means±SEM) of appropriate control value (100%).
Comparison of mean values was performed by one-way analysis of variance (ANOVA).
Significant differences (*) were assumed for p<0.05, highly significant (**) for p<0.01 and very
highly significant (***) for p<0.001.
RESULTS
Concentrations of LH, E2, and P4 in plasma of experimental gilts
Mean LH, E2, and P4 plasma concentrations in gilts of Group 1 (OVX) were
1.55±0.12 ng/ml, 14.88±0.56 pg/ml, and 0.74±0.04 ng/ml, respectively.
Treatment of OVX gilts with EB on 30-36 (Groups 2) and 60-66 h before
slaughter (Groups 3) increased (p<0.01 vs. OVX gilts) plasma E2 concentrations
to 28.17±1.55 and 21.40±0.97 pg/ml, respectively. In groups representing the
negative (Group 2) and positive (Group 3) feedback phases, the plasma LH
concentrations were 0.56±0.05 (p<0.01 vs. OVX gilts) and 2.11±0.34 ng/ml,
respectively. Progesterone administration to gilts of Group 4 increased the steroid
plasma concentration (9.89±0.25 ng/ml; p<0.01 vs. OVX gilts) to values
observed during luteal phase of the estrous cycle.
The effects of GnRH, OT and VIP on LH secretion in vitro by porcine
pituitary cells
Basal releases of LH from cultured pituitary cells of OVX gilts primed with
EB at 30-36 h (90.8±7.1 ng/ml) and 60-66 h before slaughter (112.8±5.6 ng/ml)
were significantly reduced (p<0.001) in comparison with that of placebo
receiving OVX gilts (161.6±12.4 ng/ml). Priming of OVX gilts in vivo with P4
was without effect on subsequent LH release in vitro (185.6±15.2 ng/ml).
The addition of GnRH to cultured pituitary cells resulted in marked increases
of LH (p<0.001) in all experimental groups (Fig. 1A-D). In the presence of OT
(all doses tested) and VIP (all doses) we noted significant increases (p<0.001) in
LH secretion by pituitary cells derived from gilts representing the positive
feedback phase (Group 3) (Fig. 1C). In contrast, OT and VIP (all doses) were
without any effect on LH release in Group 1 (OVX) and Group 2 (OVX+EB I;
the negative feedback phase) (Fig. 1A-B). Pituitary cells obtained from OVX gilts
444
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Fig. 1. Effects of GnRH (100 ng/ml), OT (10-1000 nM), and VIP (1-100 nM) on LH release by cultured pituitary cells (106/well) derived from gilts:
A) OVX (n=8), B) OVX+EB I (n=8), C) OVX+EB II (n=8) and D) OVX+P4 (n=9). Data are presented as the percentage (mean±SEM) of control LH
secretion by the cells (=100%). Significant differences vs. control: *** p<0.001.
0
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LH (% of control)
LH (% of control)
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primed with P4 (Group 4) produced significantly higher amounts (p<0.001) of LH
only after an addition of 100 nM OT (Fig. 1D).
The effects of GnRH, OT and VIP on PRL secretion in vitro by porcine
pituitary cells
Basal PRL secretions by pituitary cells obtained from OVX gilts primed with
placebo (Group 1), EB I (Group 2), EB II (Group 3), and P4 (Group 4) were
542.4±46.1, 450.8±39.9, 550.2±47.7, and 497.3±46.6 ng/ml, respectively.
Treatments of OVX gilts in vivo with steroid hormones, EB (Groups 2 and 3) or
P4 (Group 4), were without any effect on PRL secretion in vitro by isolated
pituitary cells.
Neuropeptide GnRH did not affect PRL secretion by pituitary cells obtained
from gilts of all experimental groups (Fig. 2A-D). Oxytocin (all doses) also failed
to alter PRL secretion in Group 1 (OVX) and Group 2 (OVX+EB I) (Fig 2A-B).
However, pituitary cells from animals primed with EB 60-66 h before slaughter
(Group 3) and P4 (Group 4) produced markedly increased amounts of PRL in the
presence of OT (Fig. 2C-D). Neuropeptide VIP stimulated PRL release from
pituitary cells of OVX gilts primed with either EB (Groups 2 and 3) or P4 (Group
4) (Fig. 2B-D). In contrast, in OVX gilts primed with placebo (Group 1), VIP was
without any effect on PRL secretion (Fig. 2A).
DISCUSSION
In the present in vitro studies, GnRH stimulated LH release from pituitary
cells of all experimental gilts and had no effect on PRL secretion. Therefore, our
results confirm that GnRH is a key factor in the regulation of LH secretion by
pituitary gland what has been established in earlier studies (2, 3, 36). Although
GnRH do not directly affect lactotroph cells (37, 38), it stimulates PRL secretion
in the presence of gonadotrophs, and intensity of the stimulation depends on their
number in the culture (38). It was demonstrated that GnRH only slightly
influences adult rat pituitary cells, but markedly stimulates PRL release from
pituitaries of neonatal and 14 days old rats (39). It should be mentioned that
pituitaries from adult individuals contain small quantity of gonadotrophic cells
(37, 38). Gonadotrophs of young and adult rats begin to respond in vitro to GnRH
after 4 and 12 days of culture, respectively (38). This difference is presumably
associated with the fact that co-operation between various types of pituitary cells
is established quicker in young than adult animals. Collectively, above
observations may suggest that GnRH-dependent PRL release from pituitary cells
under in vitro conditions requires sufficient duration of culturing and the presence
of gonadotrophs, which presumably modulate PRL secretion through their
product(s) in a paracrine fashion. Thus, in our experiment lack of GnRH effect on
446
160
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80
60
40
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120
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80
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80
60
40
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120
0
120
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PRL (% of control)
PRL (% of control)
60
G
nR
40
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nR
20
0
180
160
140
120
100
80
60
40
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10
10
0
10
00
B
D
Fig. 2. Effects of GnRH (100 ng/ml), OT (10-1000 nM), and VIP (1-100 nM) on PRL release by cultured pituitary cells (106/well) derived from gilts:
A) OVX (n=7), B) OVX+EB I (n=8), C) OVX+EB II (n=7) and D) OVX+P4 (n=5). Data are presented as the percentage (mean±SEM) of control LH
secretion by the cells (=100%). Significant differences vs. control: * p<0,05; ** p<0,01; *** p<0.001.
0
20
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P
10
0
10
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PRL (% of control)
PRL (% of control)
447
PRL secretion may result from too short incubation of the cells (3.5 h) with the
factors tested.
In the present study we have observed stimulatory effects of OT on LH and
PRL secretions in vitro by pituitary cells dependently on hormonal status of gilts.
Oxytocin significantly increased both LH and PRL secretions by pituitary cells of
OVX gilts pre-treated with P4 (Group 4) or EB 60-66 h before slaughter (Group
3). However, this peptide appeared to be ineffective in relation to the cells of
OVX gilts receiving placebo or primed with EB 30-36 h before slaughter (Group
2). These results suggest a contribution of ovarian steroids in developing the
responsiveness of the anterior pituitary to OT. Furthermore, a comparison of the
results of animal priming with EB in Groups 2 and 3 implies that estradiol
requires longer time to enable OT action on LH and PRL secretion. The role of
ovarian steroids in this regulation may be connected with an augmentation of
OTR density in target cells, including those in the pituitary. Estrogens were found
to increase OTR gene expression (40) and number of OT specific binding sites in
diverse tissues (40, 41).
Evident response of LH to OT in gilts representing the positive feedback
phase, observed in our study, indicates participation of OT in the control of the
preovulatory LH surge in pigs. This statement is in accordance with data
pertaining to the rat females. In proestrous rats, treatment with OT advanced the
LH surge and ovulation (42), but OT antagonist administration prevented the
LH surge appearance (42, 43). Moreover, on the basis of our results, we can
suppose that OT is also implicated in the regulation of periovulatory release of
PRL (32). Many reports (5, 44, 45) have documented a stimulation of PRL
release from rat pituitary cells by OT. Johnston and Negro-Vilar (43) provided
evidence that OT participates in initiation of the proestrous surge of PRL. Thus,
aforementioned observations allow to associate physiological periovulatory
increases in OT secretion (46) with an appearance of the preovulatory LH and
PRL surges in sows.
Our present study demonstrated that another tested factor, VIP, may also be
involved in the control of LH and PRL secretion in sows dependently on
hormonal status of the females. Stimulatory effect of VIP on LH secretion from
cultured porcine pituitary cells was shown only in the group of OVX gilts
mimicking the positive feedback phase between estradiol and LH levels. Reports
pertaining to the effect of VIP on LH secretion in vivo and in vitro in other
species (predominantly in rats) are contradictory. It was found that intravenous
administration of VIP did not change LH secretion. Single injection of VIP into
third ventricle led to increase of plasma LH concentration (47), but continuous
infusion of the peptide inhibited pulsatile LH secretion (48). In studies
performed in vitro, Baranowska et al. (49) have demonstrated stimulatory
influence of VIP on LH release from cultured pituitary cells of diestrous rats.
However, Ogwuegbu et al. (20) observed elevated LH secretion in response to
VIP only when the complex of goat pituitary and MBH, but not the pituitary
448
alone, was perifused. Like in our studies, VIP did not induce any changes in LH
secretion by pituitaries of OVX rats (50). The present study indicates that VIP,
effectively stimulating LH secretion from pituitary cells representing the
positive feedback phase, may act in sows as an important factor modulating the
preovulatory LH surge at the pituitary level.
Our results also confirmed that VIP may be a stimulator of PRL secretion from
porcine pituitary cells. It increased PRL secretion by the cells of all animals
primed with EB or P4 in contrast to OVX gilts pre-treated with placebo.
Numerous in vivo and in vitro experiments in various mammalian species,
including human (51), monkey (52), goat (20), and rat (49) demonstrated
stimulatory effects of VIP on PRL secretion. Collectively, this study and others
indicate that ovarian steroid milieu is required to create and maintain the
reactivity of pituitary cells to VIP.
The mechanism of OT and VIP influence on secretion of pituitary hormones
is not entirely explained. These peptides may certainly exert direct effects on
lactotrophs, which posses both OT (41) and VIP (53) receptors. So far, the
presence of receptors for OT (54) and VIP (55) on gonadotrophs has been only
suggested. The question, whether OT and VIP affect directly or indirectly LH
secretion at the pituitary level, needs to be elucidated.
In summary, the results of our in vitro studies confirmed the stimulatory effect
of GnRH on LH secretion by porcine pituitary cells and also suggest a
participation of OT and VIP in modulation of LH and PRL secretion at the
pituitary level in a way dependent on hormonal status of animals.
Acknowledgements: We are grateful to Dr. B. Szafranska for advice regarding the method of
pituitary cell culture; Dr. M. Koziorowski and Dr. J. Staszkiewicz for help in surgical preparation
of experimental animals and to Mrs. M. Ledwozyw and Dr. I. Biallowicz for their technical
assistance. This research was supported by the State Committee for Scientific Research (project
02040.205).
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R e c e i v e d : February 11, 2002
A c c e p t e d : July 11, 2002
Author’s addres: Department of Animal Physiology, University of Warmia and Mazury in
Olsztyn, 10-718 Olsztyn-Kortowo 5, Poland. fax: +48 89 5233937
E-mail: [email protected]