Bull Vet Inst Pulawy 49, 411-417, 2005
POLYCHLORINATED BIPHENYLS IMPAIR FSH
- STIMULATED EFFECT ON THE SECRETION
OF STEROIDS AND OXYTOCIN
FROM CULTURED BOVINE GRANULOSA CELLS
JAROSŁAW J. MŁYNARCZUK1, ALICJA NIEWIADOWSKA2 AND JAN KOTWICA1
1
Institute of Animal Reproduction and Food Research, Polish Academy of Science, 10-718 Olsztyn
2
National Veterinary Research Institute, 24-100 Pulawy, Poland
e-mail: [email protected]
Received for publication September 28, 2005.
Abstract
Follicular fluid (FF) was collected by aspiration from
bovine follicles at slaughterhouse and PCBs (IUPAC number
28, 52, 101, 118, 153, 138, and 180) were determined by gas
chromatography. Total amount of PCB never exceeded 0.11
ng/ml of FF, thus we assume the granulosa cells we used in the
further studies were not influenced by PCBs. In next
experiment the effect of PCB 126, 77 or 153 (each at the dose
1, 10 i 100 ng/ml) for 72 h on the secretion of oestradiol (E2),
progesterone (P4) or oxytocin (OT) from non-stimulated or
stimulated with FSH (100 ng/ml for last 24 h) granulosa cells
(2x105/ml), obtained from bovine follicles >1< cm in diameter.
Each of the used congeners could reduce (P<0.05) effect of
FSH on E2 secretion and influence by PCB 153 in follicles of
<1cm in diameter was the most evident (P<0.05). All used
PCBs, but PCB 153 in cultures of granulosa cells from
follicles >1cm decreased the effect of FSH on the secretion of
P4. Each of used congeners stimulated OT secretion from
granulosa of both sizes follicles and PCB 77 was the most
efficient in this case (P<0.01-0.001). Data suggest that PCBs
can impair growth and development of follicle by blockade of
FSH effect on granulosa cells. Moreover, glucocorticoid and
SF-1 receptors are suppose to be also involved in PCBs
influence on OT secretion by bovine granulosa cells.
Key words: cows, granulosa cells, PCBs,
progesterone, oestradiol, oxytocin.
Polichlorinated biphenyls (PCBs) were widely
used in the industry in past time (10). Due to their
resistance for the degradation (3, 21) and penetration to
the food chain (5, 43) they are recognized as
environmental pollutants, which can be accumulated in
the tissue of humans and animals (2, 7). It was proved
that PCBs present in living bodies can mimic the effect
of oestradiol (E2; 32, 42) and they can also be bound by
arylhydrocarbon receptors (AhR) (29, 34, 35). As an
effect they harm steroidogenesis in the ovary (1, 41),
and adrenals (11), they can also accelerate metabolism
of oestradiol (4) and this way impair both the
development and function of the reproductive tract (6,
30). Moreover, individual PCBs and their mixture Aroclor 1248 stimulates bovine granulosa cells for the
secretion of oxytocin (OT) (J. Młynarczuk and J.
Kotwica – unpublished data), which plays a crucial role
in the ovarian steroidogenesis in mammals (37). Follicle
stimulating hormone (FSH) is the main regulator of E2
synthesis in granulosa cells, but this gonadotropin is also
involved in the secretion of OT from granulosa cells
(38).
Therefore the aim of the present studies was to
investigate the effect of PCBs on the secretion of E2,
progesterone (P4), and OT from granulosa cells obtained
from the follicles of two sizes. Three PCB congeners
were used: PCB 126 which has dioxin-like properties
and acts via AhR (22), PCB-77 which is both oestrogenlike (26) and has also an affinity to AhR (29, 35), and
PCB 153 which has an affinity to oestrogen receptor
mainly (33).
Material and Methods
Collection of follicular fluid. Follicular fluid
was collected at slaughterhouse by puncture of follicle
with syringe and 10 gauge needle from 30 cows. Fluid
was immediately places in tube and chilled on ice-bath.
After transportation to the laboratory within 1 h,
follicular fluid was centrifuged for 10 min at 800 x g and
samples were frozen until analysis.
Gas chromatography. Levels of chlorinated
hydrocarbons were determined by capillary gas
chromatography. Congeners of PCB (PCB IUPAC
number 28, 52, 101, 118, 153, 138, and 180) were
extracted from the follicular fluid from one cow or from
it was combined from 2-4 cows, using mixture of
hexane and acetone. The extracts were cleaned up on the
basis of sulfuric acid treatment (H2SO4 + H2SO4 25%
SO3), while separation of PCB congeners from
organochlorine pesticides was achieved using alkaline
412
hydrolysis (10% KOH in ethanol). The purified extracts
were analysed by GLC-ECD. Gas chromatographic
analysis was performed on HP 6890 Plus equipped with
63
Ni-µEC detector and split-splitless injector in pulsed
splitless mode. Analytical fused silica capillary column
HP – 5MS 60 m x 0.25 mm i.d. with film thickness 0.25
µm was used. Temperature for the column was
programmed: 60°C (1.5 min) 25°C/min 150°C (2 min)
2.5°C/min 275°C (17.9 min), run time 75 min. Injector
and detector temperatures were set at 280°C and 300°C,
respectively. Helium and nitrogen were used as carrier
and makeup gases. The research was based on a
validated analytical method in accredited laboratory.
The limit of determination was 0.05 ng/g and recoveries
from fortified samples exceeded 85%. In addition, the
certified reference materials were analysed.
PCBs 138 and 180 were the most common
measured in the follicular fluid (from non-detected value
to 0.08 ng/ml). Total amount of PCBs was never higher
than 0.11 ng/ml. Therefore we conclude that granulosa
cells we used in our studies were not affected by PCBs
and we could measured a pure reaction of cells on PCBs
treatment.
Collection of ovaries. Bovine ovaries from
cows or mature heifers at defined stage of the oestrous
cycle (days 6-16 for the follicles <1 cm, and 16-21 for
follicles >1 cm), as classified according to Ireland et al.
(14) and Fields and Fields (8), were collected from a
commercial slaughterhouse within 15-20 min after
killing the animals. The ovaries were placed in an icecold 0.9% NaCl containing penicillin (10 IU/ml),
streptomycin (100 µg/ml), amphotericin (2 µg/ml), and
L-glutamine (100 µg/ml) and then transported to the
laboratory on ice-bath within 1 h. Atretic follicles were
eliminated on the basis of examinations given by
Henderson et al. (13). All materials used were purchased
from Sigma (Poznan, Poland) unless otherwise stated.
Collection of granulosa cells. Granulosa cells
were collected by vigorous aspiration of follicular fluid
(38) separately from the follicles of two sizes (above and
below 1 cm in diameter). Suspension of cells was
collected in tube stored on ice-bath. To collect the cells
for one plate, 8-10 follicles >1 cm or 12-14 follicles <1
cm, from the same stage of the oestrous cycle were used.
Cells were washed twice with M-199 containing Earle’s
salt and 0.1% BSA and suspended in DMEM/F-12
HAM enriched with 10% FCS. Viability of the cells,
estimated by exclusion of trypan blue (0.04%) dye, from
follicles <1 cm of diameter was 65-75% and about 75%
for cells from follicles >1 cm of diameter. Next,
suspension of the cells (2x105/ml) was transferred (0.5
ml) into 48-well plate (Nunclon, NUNC). After 24 h
preincubation they were washed twice with M-199
containing 0.1% BSA and suspended in 0.5 ml of
DMEM/F-12 HAM/0.1% BSA supplemented with
ascorbic acid (20 µg/ml), transferrin (5 µg/ml) and
sodium selenite (5 ng/ml, ICN). Next, cells were
cultured in atmosphere of air containing 5% CO2 at
100% humidity, and at 38oC (Heraus BB-6060, Hanau,
Germany). All media were enriched with gentamycin
(20 µg/ml; ICN Pharmaceuticals, Inc., Costa Mesa,
USA).
Experiment 1. Granulosa cells from follicles
>1 and <1 cm of diameter were treated with 1, 10 or 100
ng/ml
either
of
PCB
126
(3,3’4,4’,5pentachlorobiphenyl),
PCB
77
(3,3’,4,4’tertrachlorobiphenyl) or PCB 153
(2,2,4,4’,5,5’hexachlorobiphenyl). The FSH (100 ng/ml; 38) was
given to the media on the last 24 h of incubation.
Hydrocortisone (10-5M) was used as positive control
(24) for OT secretion. Medium was collected after 72 h
of incubation and stored at –200C before hormone
analysis. Each treatment was used in 6-8 replicates.
Experiment 2. To verify the hypotheses that
PCBs can influence the cell function via glucocorticoid
receptor, granulosa cells from follicles >1 cm of
diameter were incubated for 72 h with 1, 10 or 100
ng/ml of PCB 77 or 153 and each of them jointly with
RU 486 (10-5M) which is blocker of glucocorticoid
receptor.
Hormone assays. The concentration of E2 in
the culture media were determinated by EIA. Antioestradiol serum (gift from Dr G.L. Williams) was used
at final dilution of 1:150 000. Cross-reaction of these
antibodies with estrone, 17α-oestradiol and oestradiol
benzoate were 9.0, 2.2 and 70.8%, respectively. The
range of curve was 6.25-1 600 pg/ml and sensitivity of
method was 20-25 pg/ml. Intra- and inter- assay
coefficients of variation were on average 10.8% and
12.4%. Accuracy and reliability of the procedure was
expressed by a coefficient of regression of r=0.889. A
reader plate (Multiscan EX, Labsystem, Finland) was
used for the measurement of absorbance at 450 nm for
both E2 and two other hormones determined in these
studies.
The concentrations of OT in the culture
medium were determined by EIA as reported earlier
(36). Anti-oxytocin serum (R-1), which has been
previously characterized (19), was used at a final
dilution of 1 : 30 000. The range of the curve was 3.9-1
000 pg/ml and the sensitivity of the procedure was 18-20
pg/ml. Intra- and inter-assay coefficients of variation
were on average 9.6% and 11.4%, respectively.
Accuracy and reliability of the procedure was expressed
by a coefficient of regression of r=0.90.
Progesterone concentrations in media were
determined by EIA as described by Prakash et al. (31)
Progesterone labelled with horseradish peroxidase was
used at final dilution of 1 : 8 000, and P4 antiserum
(IFP4) used at a final dilution of 1 : 100 000 was
characterized in an earlier work (20). The range of the
standard curve was 0.1-25 ng/ml and the sensitivity of
the procedure was 0.15 ng/ml. Intra- and inter-assay
coefficients of variation were on average 8.6% and
10.7% respectively. The relationship between the added
and measured amounts (n=8) of hormone was r=0.96.
Statistical analysis. Mean (±SEM) concentrations of hormones within groups were compared by
ANOVA and by Tukey post-test using GraphPad
PRISM Software.
413
Results
None of the used PCBs affected significantly
basal secretion of E2 from follicles of both sizes
compared to control (Figs 1, 2). But each of them (at the
different doses) inhibited stimulatory effect of FSH on
E2 secretion (Figs 1, 2). PCB 126 most clearly
(P<0.001) inhibited the effect at the lowest dose from
the follicles of <1 cm diameter (Fig. 1), while in
granulosa cells from larger follicles (<1 cm) all doses of
this congener inhibited the effect of FSH (P<0.05–0.01;
Fig. 2). The effect of FSH was also inhibited by PCB 77
in dose-dependent manner (P<0.05–0.001). Oestrogenlike PCB 153 blocked (P<0.001) FSH effect in the cells
from small follicles (<1 cm) but in the cells from the
follicles of >1 cm diameter this congener at the dose of
100 ng/ml only abolished stimulatory influence of FSH
750
b
750
(P<0.05). Control granulosa cells from the follicles of
both sizes secreted a similar amount of E2. In contrast,
P4 and OT were secreted in higher concentrations from
granulosa cells obtained from the larger follicles.
Hormone secretion after treatment of cells with PCB
was proportional to their basic secretion.
Basic secretion of P4 from granulosa cells of
follicles of both sizes was changed by none of the used
PCBs (P<0.05) (Figs 3, 4). However, each of the
congeners at different doses (PCB 126: 10, 100 ng/ml;
PCB 77: 1, 100 ng/ml; all doses of PCB 153) inhibited
stimulatory effect of FSH from follicles of <1 cm
diameter (P<0.05 – 0.01; Fig. 3), while PCBs 126 and
77 in all used doses, but none of PCB 153, diminished
(P<0.05 – 0.01) stimulatory effect of FSH on P4
secretion from cells obtained from follicles of >1cm
diameter.
bc
abc
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ac
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10
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Concentrations of E2 (pg/ml)
100
c
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250
Ctrl FSH
1
10 100
PCB 77
1
10 100
PCB 77+FSH
a
10
100
a
a
PCB 126
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10
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PCB 126+FSH
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PCB 77
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PCB 77+FSH
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Ctrl FSH
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Concentrations of E2 (pg/ml)
Ctrl FSH
PCB 126
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Ctrl FSH
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PCB 153
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10
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PCB 153+FSH
Fig. 1. Influence of PCBs (1, 10, 100 ng/ml), acting for
72 h with or without FSH (100 ng/ml), on mean
(±SEM) secretion of oestradiol (E2) from bovine
granulosa cells obtained from follicles of <1 cm in
diameter. Bars with different superscripts are different
(P<0.05).
0
Ctrl FSH
1
10
100
PCB 153
1
10
100
PCB 153+FSH
Fig. 2. Influence of PCBs (1, 10, 100 ng/ml), acting for
72 h with or without FSH (100 ng/ml), on mean
(±SEM) secretion of oestradiol (E2) from bovine
granulosa cells obtained from follicles of >1 cm in
diameter. Bars with different superscripts are different
(P<0.05).
414
30
40
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20
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PCB 126
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PCB 126+FSH
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b
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ad
ad
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ac
a
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cd
a
10
Ctrl FSH
1
10
100
1
10
Concentrations of P4 (ng/ml)
Concentrations of P4 (ng/ml)
1
PCB 126
40
0
Ctrl FSH
PCB 126+FSH
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30
ad
ad
Ctrl FSH
1
20
0
a
10
100
cd
1
10
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PCB 77+FSH
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PCB 77
PCB 77+FSH
PCB 77
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100
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ac
40
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20
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Ctrl FSH
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PCB 153
1
10
100
PCB 153+FSH
Fig. 3. Influence of PCBs (1, 10, 100 ng/ml), acting for 72
h with or without FSH (100 ng/ml), on mean (±SEM)
secretion of progesterone (P4) from bovine granulosa
cells obtained from follicles of <1 cm in diameter. Bars
with different superscripts are different (P<0.05).
Effect of PCB 77 and 153 on OT secretion was
significantly (P<0.05-0.01) reduced by blocker of
glucocorticoid receptor (Fig. 7).
All used PCB congeners, except the lowest
dose of PCB 126 and 153, stimulated (P<0.05-0.001)
OT secretion from granulosa cells obtained from the
follicles of <1 cm of diameter (Fig. 5). The most
effective stimulator was PCB 77. Concomitantly, this
congener markedly (P<0.01) inhibited effect of FSH
(Fig. 6), while influence by two other congeners, though
evident, was less pronounced. All congeners stimulated
also OT secretion from granulosa cells of larger follicle
(>1 cm), and again effect of PCB 77 was the most
evident (P<0.001). Except dose of 100 ng/ml of PCB
153, PCBs used at all other doses inhibited (P<0.050.001) FSH-stimulated OT secretion.
Discussion
In milk of cows in Poland PCBs concentration
reached 0.4 ng/ml (43), while in follicular fluid it fluc-
0
Ctrl FSH
1
10
100
PCB 153
1
10
100
PCB153+FSH
Fig. 4. Influence of PCBs (1, 10, 100 ng/ml), acting for 72
h with or without FSH (100 ng/ml), on mean (±SEM)
secretion of progesterone (P4) from bovine granulosa
cells obtained from follicles of >1 cm in diameter. Bars
with different superscripts are different (P<0.05).
-tuated from non-detectable concentrations up to 110
pg/ml in individual cow. Therefore, the experimental
effects we observed were not influenced by previous
effect of PCBs, while the doses of PCB we have used
were within the range of that measured in the follicular
fluids in ruminants from other countries (18). There was
a slight, if any, direct effect of PCBs on E2 and P4
secretion from bovine granulosa cells. This may suggest
that PCBs do not affect steroidogenesis in this
experimental model, as we found using luteal cells (25).
Or there are some differences between species, since
PCBs were found to affect E2 secretion in swine (41).
However, each of the used PCB inhibited stimulatory
effect of FSH on E2 secretion, likely to the results
obtained by Nikula et al. (28) who found inhibitory
effect of bisphenol A and octophenols in mLTC-1 cell
line. Since FSH increases the activity of aromatase in
granulosa cells (9), therefore it can be assumed that
PCBs interferes in some way in the mechanism of E2
synthesis/secretion, supposedly via the decline in the
intracellular concentrations of cAMP (28).
415
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Ctrl FSH Cort
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10 100
PCB 126
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10 100
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Ctrl FSH Cort
Ctrl FSH Cort
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10 100
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10
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75
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d
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Ctrl FSH Cort
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PCB 77
PCB 77 + FSH
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75
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b
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d
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ac
25
100
PCB126 + FSH
c
PCB 77+FSH
PCB 77
10
PCB 126
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Concentrations of OT (pg/ml)
1
PCB 126+FSH
Concentrations of OT (pg/ml)
0
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de
ce
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50
a
c
cd
c
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a
25
0
Ctrl FSH Cort
1
10 100
PCB 153
1
0
10 100
Ctrl FSH Cort
Fig. 5. Influence of PCBs (1, 10, 100 ng/ml), acting for
72 h with or without FSH (100 ng/ml), on mean
(±SEM) secretion of oxytocin (OT) from bovine
granulosa cells obtained from follicles of <1 cm in
diameter. Bars with different superscripts are different
(P<0.05).
Concentrations of OT (pg/ml)
10
100
1
10
100
PCB 153+FSH
Fig. 6. Influence of PCBs (1, 10, 100 ng/ml), acting for
72 h with or without FSH (100 ng/ml), on mean
(±SEM) secretion of oxytocin (OT) from bovine
granulosa cells obtained from follicles of >1 cm in
diameter. Bars with different superscripts are different
(P<0.05).
b
100
bc
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50
c
d
a
ad
a
a
1
10
25
0
Ctrl Cort
1
10
100
PCB 77
100
PCB 77+RU486
b
100
Concentrations of OT (pg/ml)
1
PCB 153
PCB 153+FSH
b
b
75
c
50
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ac
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a
25
0
Ctrl
Cort
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10
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PCB 153
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10
100
PCB 153+RU486
Fig. 7. Effect of RU486 on mean (±SEM) secretion of OT by granulosa cells obtained from follicles >1 cm after treatment (72 h)
with PCB 77 or 153. Bars with different superscripts are different (P<0.05-0.001).
416
It was showed earlier that small doses of E2 can
stimulate OT secretion from bovine granulosa cells (39).
Thus PCB 77, which has low affinity to E2 receptor (26,
27) but was used in high amount in this study, could
elicit E2 effect on OT secretion as we have found in the
present studies (Fig. 5). However, it can be assumed that
E2 receptor is not the only involved in PCBs-stimulated
OT secretion, since granulosa cells stimulated with E2
(10-9M) secreted less OT (P<0.05) compared to PCBs
used in present studies (J. Młynarczuk and J. Kotwica –
unpublished data). This suggestion can be supported by
data that dioxin-like congeners are antagonists of E2
receptor (12, 15), though PCB 126 could increase OT
release (Figs 5, 6). Moreover, some increase in OT
release was evoked by the mixture of PCBs – Aroclor
1248 (J. Młynarczuk and J. Kotwica – unpublished
data). Further, OT secretion was also stimulated by the
activation of glucocorticoid receptors as found earlier
(24) and by orphan receptor SF-1 which increases
expression of gene for neurophysin/oxytocin (40). These
results are in agreement with our data (Fig. 7) that
RU486 (blocker of glucocorticoid receptors) diminished
stimulatory effect of PCBs on OT secretion by granulosa
cells.
The biphasic effect of FSH on the secretion of
OT by granulosa cells has been suggested (38). During
follicular growth FSH increases OT release from
granulosa cells, but just before ovulation it stops OT
secretion and concomitantly increases P4 release (16).
Similar effect of FSH was observed in these studies but
this effect was diminished by PCBs. These observations
could explain the disruptive influence of these
compounds on the development of follicle. Since
increased concentration of OT in the follicular fluid
leads to the premature luteinization of granulosa cells
(17, 23) in the immature follicles, and further it declines
cell response to FSH stimulation. Hence, it is possible
that effect of PCBs upon reproductive processes takes
place via direct influence on AhR and receptors for E2
and next, this decreases the intracellular concentration of
cAMP as suggested by Nikula et al. (28). So, this way
PCBs can impair a vital reproductive processes (eg.
follicles development). Therefore it can be hypothesized
that PCBs decline the effect of FSH on E2 and OT
secretion from granulosa cells in two ways: (i) they
reduce intracellular concentration of cAMP and thus
disrupt signal transmission from receptor and (ii) PCBs
can affect also the follicular synthesis/secretion of OT
and this hastens luteinization of steroidogenic cells.
In conclusion, the data suggest that PCBs block
FSH effect on granulosa cells and can impair growth and
development of follicle. Moreover, except AhR and E2
receptors, glucocorticoid and SF-1 receptors are suppose
to be also involved in the mechanism of PCBs influence
on OT secretion by granulosa cells in cow.
Acknowledgments: We thank Drs G. Kotwica
and S. Okrasa (University of Warmia and Mazury,
Olsztyn, Poland) for the antibodies against oxytocin and
progesterone, and Dr G.L. Williams (A&M Texas
University, College Station, USA) for antibody against
oestradiol-17β. Studies were supported by Solicited
Project from KBN (PBZ-KBN-084/P06/2002) and by
the Polish Academy of Sciences.
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