185
Vol. 4, No. 2
SHORT COMMUNICATION
Genistein affects testosterone
secretion by Leydig cells in roosters
(Gallus gallus domesticus)
Marek Opałka, Barbara Kamińska, Renata Ciereszko, Luiza Dusza
Department of Animal Physiology, University of Warmia and Mazury in
Olsztyn, Poland
Received: 4 May 2004; accepted: 19 June 2004
SUMMARY
Genistein is one of non-steroidal phytoestrogens present in soya and
soybean products as well as in other legumes. Phytoestrogens possess
estrogen-like biological activity and may influence human and animal
reproduction. The aim of this study was to examine the effect of genistein on testosterone (T) secretion by isolated Leydig cells in roosters.
Genistein (5-50 μM) inhibited (p<0.05) in vitro basal and LH-stimulated
T secretion by Leydig cells in a dose dependent manner. No significant
effect of lavendustin C (inhibitor of PTK, a non-phytoestrogen) on the T
production was observed. In conclusion, genistein, present in commercial poultry feeds, may influence testicular steroidogenesis but its effect
on reproductive performance of roosters requires further examinations.
Reproductive Biology 2004 4 (2): 185-193.
Key words: phytoestrogens, genistein, testis, male domestic fowl,
testosterone
Corresponding author: Department of Animal Physiology, University of Warmia and Mazury in
Olsztyn, Oczapowskiego 1A, 10-718 Olsztyn, Poland; e-mail: [email protected]
1
Copyright © 2004 by the Society for Biology of Reproduction
186
Genistein and steroidogenesis in roosters
INTRODUCTION
Genistein is a non-steroidal phytoestrogen, present in soya and soybean products as well as in other legumes, constituents of the most commercial poultry
feeds [22]. The phytoestrogens, possessing estrogen-like biological activity,
have been found to influence human and animal reproduction by changing
sexual behaviour as well as morphology and function of reproductive organs
[for review see: 19]. In addition, genistein, but not other phytoestrogens,
has been described as a specific inhibitor of tyrosine protein kinases (PTK;
[2]). However, only few studies concerning dietary phytoestrogen effects
on avian reproduction have been performed. California quail fed a diet with
a high concentration of phytoestrogens (genistein, formononetin) had poor
breeding success [15]. No effect on reproductive performance was observed
in bobwhites which were fed with biochanin A [16].
Several effects of genistein on ovarian [5, 6, 17, 27] and adrenal [10,
18] steroidogenesis have been reported in mammals. Studies performed on
testicular cell lines showed that genistein inhibited the growth and proliferation [14] as well as induced apoptotic cell death [13]. Since there is no data
concerning phytoestrogens and testicular steroidogenesis in domestic fowl,
the aim of the study was to examine the effect of genistein on testosterone
(T) secretion by isolated Leydig cells in roosters.
MATERIALS AND METHODS
All experiments were performed in accordance with the principles and
procedures of the Animal Ethics Committee of the University of Warmia
and Mazury in Olsztyn. Six, sexually matured Cobbe 500 line roosters (21
week old, 3.64±0.10 kg body weight) originated from commercial farm.
The animals were euthanized by decapitation. Testes were removed and
transported to the laboratory in ice cold Ham’s F-12 medium.
Testes were decapsulated and minced. The Leydig cells were dispersed
by grinding through the 80 μm pore-size steel sieve (Cell Dissociation
Sieve-Tissue Grinder Kit, Sigma, Germany). The purity of cells (48-53%)
was examined by a histochemical test for 3β-hydroxysteroid dehydrogenase
Opałka et al.
187
activity [3, 23]. The isolated cells were washed twice in Ham’s F-12 medium and centrifuged (150 x g, 10 min). Then the steroidogenic cells were
counted by haemocytometer, diluted to density of 2.5x105/ml and seeded
(1 ml) into 24-well culture plates. Ham’s F-12 containing 2% bovine serum
albumin and supplemented with 25 mM Hepes buffer, 1.176 g/L NaHCO3
and antibiotics was used as an incubation medium. The cells were incubated
for 6 h with or whitout LH (2 or 20 ng/ml), genistein (0.5, 5, 10 or 50 μM)
and lavendustin C (0.5, 5, 10 or 50 μM). The concentration of treatments
and time of incubation were established in our preliminary studies. Incubations were performed under a water-saturated atmosphere of 95% air and
5% CO2 at 37°C. After the end of incubation, media were collected and
stored at −20°C until determination of T concentration.
Radioimmunoassay (RIA) was used to measure concentrations of T
in samples of medium incubated with dispersed rooster Leydig cells. The
validations of this assay for use in experiments with porcine ovarian cells
[4] as well as fish plasma [8] have been reported previously. In order to
validate the assay for use with medium incubated with domestic fowl Leydig
cells, a standard curve with a slope of approximately –1.0 after a log/logit
transformation was established using 1- 200 pg standards. T antiserum was
characterized elsewhere [24]. Exogenous treatment did not interfere with the
RIA. Sensitivity of the assay was 5 pg per tube. Intra-assay and inter-assay
coefficients of variation were 1.7% and 2.4%, respectively. Radiochemical
was purchased from Amersham (UK) and pLH was provided by A.F. Parlow
(NHPP). All other reagents were purchased from Sigma (Germany). All data
are presented as mean±SEM. Log-transformed data were submitted to one
way analysis of variance for repeated measurements followed by LSD-test
(Statistica, StatSoft Inc., Tulsa OK, USA).
RESULTS AND DISCUSSION
Genistein (5, 10 and 50 μM) inhibited (p<0.05) T secreted by Leydig
cells in a dose dependent manner (fig. 1). Secretion of T was stimulated
by 2 ng (p<0.01) and 20 ng (p<0.05) of LH (fig.2). The LH-stimulated
T secretion was decreased (p<0.05) by three out of four doses of ge-
Genistein and steroidogenesis in roosters
188
140
c
cb
ab
Testosterone (pg/ml)
120
a
a
100
80
60
40
20
0
Control
0.5
5
10
50
Genistein (µM)
Fig. 1. The effect of genistein on testosterone (T) secretion (mean±SEM) by rooster
Leydig cells (n=6 independent experiments). Cells (2.5×105) were incubated for 6 h
(37°C). Bars with the same superscripts are not significantly different (p<0.05).
nistein (5-50 μM). Similar, hollyhock extract (Althaea rosea Cav. Var.
nigra) containing other phytoestrogens - quercetin and kaempferol
- decreased Leydig cells secretion of T in rats [21]. The effect of phytoestrogens on avian steroidogenesis and/or reproduction, however,
has not been extensively examined. An inverse correlation between
the incidence of estrogenic substances (genistein, formononetin) in
the diet and reproductive success was observed in the quail [15]. In
addition, high dose (1 mg/day) of biochanin A was found to impair
reproduction in bobwhites [16].
In fish (Oryzias latipes), genistein injection resulted in a decreased T
production from ex vivo testes and a comparable reduction in circulating T
level [29]. Feeding of the phytoestrogen-rich diet (600 μg isoflavones/g of
diet) decreased plasma T and androstenedione in male rat [26]. Exposure
of female rats to dietary genistein during gestation and lactation resulted
in a lower plasma T concentration in their adult male offspring [28]. Thus,
phytoestrogens exert adverse effects on reproduction and steroidogenesis
in males of different vertebrates.
Opałka et al.
Testosterone (pg/ml)
250
b
200
150
189
ab
a
a
a
a
100
50
0
Genistein ( µM)
-
-
0.5
5
10
50
LH (2ng/ml)
-
+
+
+
+
+
c
Testosterone (pg/ml)
250
c
bc
200
150
ab
ab
a
100
50
0
Genistein ( µM)
-
-
0.5
5
10
50
LH (20 ng/ml)
-
+
+
+
+
+
Fig. 2. The effect of genistein on basal and LH-stimulated testosterone (T) secretion (mean±SEM) by rooster Leydig cells (n=6 independent experiments). Cells
were incubated for 6 h (37°C) in the presence or absence of 2 ng (upper panel)
or 20 ng (lower panel) of porcine LH. Bars with the same superscripts are not
significantly different (p<0.05).
On the other hand, neither serum T concentration nor sperm counts
were changed by neonatal oral administration of genistein to male rats
[20]. Similarly, dietary genistein did not affect testicular weight or mor-
Genistein and steroidogenesis in roosters
190
140
a
a
Testosterone (pg/ml)
120
a
a
a
100
80
60
40
20
0
Control
0.5
5
10
Lavendustin ( µM)
50
Fig. 3. The effect of lavendustin C on testosterone (T) secretion (mean±SEM) by rooster
Leydig cells cells (n=6 independent experiments). Cells (2.5×105) were incubated for
6 h (37°C). Bars with the same superscripts are not significantly different (p<0.05).
phology as well as androgen receptor, epidermal growth factor receptor
and extracellular signal-related kinase expression in rat testicular cells
[9]. It is possible that the effect of phytoestrogens may depend on the
species, reproductive status of the animal, length of the exposure, the
way of treatment administration (diet, injection, in vitro treatment) and
treatment level.
Genistein was reported to alter ovarian and adrenal steroidogenesis in
vitro. This phytoestrogen inhibited progesterone production by porcine theca
[6] and luteal cells [4] as well as by rat granulosa-luteal cells [27]. In our
other study, genistein suppressed basal and PRL-stimulated cortisol secretion by porcine adrenocortical cells [10]. Diets with genistein inhibited T
conversion to estradiol, a reaction catalyzed by aromatase [11]. Moreover,
phytoestrogens inhibited 17β-hydroxysteroid dehydrogenase, an enzyme
converting androstenedione to T [12]. Phytoestrogen action on target cells
appears to be associated with their estrogenic or antiestrogenic activity. In
addition to inhibition of key steroidogenic enzymes, genistein was found
to affect estrogen receptors [25].
Opałka et al.
191
Genistein is known to be a specific inhibitor of PTK [2]. Protein tyrosine
kinases are involved in signal transduction pathways of growth factors and
cytokines that are known to exert modulatory effects on steroidogenesis [7].
To test whether the inhibitory action of genistein may result from its effect
on PTK activity, we have examined a non-phytoestrogen - lavendustin C, a
potent inhibitor of PTK [1]. In contrast to results of Whitehead and Lacey
[27] who showed that lavendustin and genistein suppressed progesterone
production by rat ganulosa-luteal cells, in our study, lavendustin C did not
change T secretion by incubated rooster Leydig cells (fig. 3). This suggests
that genistein does not affect steroidogenesis of rooster Leydig cells by
inhibition the PTK system. In conclusion, genistein present in commercial
poultry feeds may influence testicular steroidogenesis in roosters. However,
further studies are required to ascertain whether the phytoestrogens may
also affect in vivo reproductive performance of these animals as well as to
examine mechanism of genistein action in animal steroidogenic cells.
ACKNOWLEDGMENTS
The study was supported by State Committee for Scientific Research, Poland (project No 0206 0804). Porcine LH was kindly provided from Dr.
A. F. Parlow, National Hormone & Peptide Program, Torrance, California,
USA.
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