Vol. 6, Suppl. 1
189
The influence of polychlorinated biphenyls (PCBs)
and phytoestrogens in vitro on functioning
of reproductive tract in cow
Jan Kotwica1, Michał Wróbel, Jarosław Młynarczuk
Institute of Animal Reproduction and Food Research,
Polish Academy of Sciences, Olsztyn, Poland
SUMMARY
Ovarian, endometrial and myometrial cells and strips of longitudinal
myometrium from cows on defined days of estrous cycle were treated
for 24-72 h with different doses (1-100 ng/ml) of PCBs mixture (Aroclor
1248) or with one of PCB congeners (126, 77, 153). The administered
doses of PCBs neither affected the viability of cells nor influenced the
ovarian steroidogenesis as measured by progesterone (P4), estradiol
(E2) and testosterone secretion from luteal, granulosa and theca cells,
respectively. In contrast, PCBs clearly inhibited a FSH and LH-stimulated
effect on steroids secretion from granulosa and luteal cells. Moreover,
PCBs significantly stimulated oxytocin (OT) secretion from the studied
ovarian cells, and at least part of this effect is elicited through activation
of glucocorticoid receptors. Further, PCBs were found to increase basal
intracellular concentrations of Ca2+ and both spontaneous and OTstimulated contractions of myometrial strips. Concomitantly, PCBs
increased endometrial secretion of PGF2α, hence the ratio of PGF2α:PGE2
was also increased. Phytoestrogens (genistein, daidzein, coumestrol),
Corresponding author: Institute of Animal Reproduction and Food Research, Polish Academy of
Sciences, 10-718 Olsztyn-Kortowo; Poland; e-mail: [email protected]
1
Copyright © 2006 by the Society for Biology of Reproduction
190
PCBs and phytoestrogens affect reproduction in cows
with a different intensity, reduced the effect of PCBs on PGF2α secretion
and myometrial contractions. Genistein inhibited PCBs’ effect on OT
secretion from granulosa cells, while PCB’s effect on OT release from
luteal cells was reduced mainly by genistein and daidzein. We conclude
that PCBs can impair both ovarian functioning and uterine contractility,
while phytoestrogens are able to reduce this effect. Reproductive Biology
2006 6 Suppl. 1:189–194.
Key words: PCBs, phytoestrogens, ovary, uterus, oxytocin, cow
Polychlorinated biphenyls (PCBs) are recognized as environmental
pollutants due to their resistance to degradation [4] as well as their ability
to be accumulated in animal tissues and to interfere with reproductive
processes e.g. premature parturition or abortion [1]. They have been found
in many tissues of human and animal reproductive tracts, e.g. in follicular
fluid [5, 14], uterus [7], and placenta [15]. PCBs can be bound within a
cell by estradiol (ER; [10]) and arylhydrocarbon (AhR; [16]) receptors.
We present some new data on the disruptive effects of PCBs in ovarian and
uterine cells on defined days of the estrous cycle. To avoid the criticism
that doses (1-100 ng/ml) of PCB mixture (Aroclor 1248) and congeners
(77, 126, 153), which have an affinity to ER or AhR, could affect the
viability of cells, this parameter was measured. It appeared that none of the
doses of PCB affected the viability of luteal, granulosa, endometrial and
myometrial cells within 48 and 72h of treatment [11, 19, 20]. However,
PCBs can affect cell viability after this time since PCB 77 (100 ng/ml)
increased expression of proapoptotic gene bax, changed the ratio of bax/
bcl-2 and also increased caspase-3 activity in luteal cells [9].
Effect of PCBs on ovarian functions
Neither Ar1248 [13] nor PCB congeners at doses of 1, 10 or 100 ng/
ml [12] affected progesterone (P4) secretion from luteal cell within 2472h. In addition, no congeners influenced the basal secretion of E2 and
P4 by granulosa cells from follicles of <1 cm and >1cm in diameter
[11]. Testosterone secretion from theca interna cells of follicles was not
Kotwica et al.
191
affected by Ar1248 and administered PCB congeners (unpublished data).
Moreover, in contrast to other species [2], Ar1248 did not impair the
utilization of high density lipoproteins [13] which are substrate for P4
synthesis in bovine luteal cells. However, PCB congeners inhibited FSHstimulated E2 and P4 secretion from granulosa cells [11]. Furthermore,
PCB 77 and 126, in contrast to Ar1248, inhibited – with different intensity
– the stimulatory effect of LH on P4 release from luteal cells on different
days of the estrous cycle [12]. This discrepancy between effects of
Ar1248 and individual congeners could be elicited due to higher toxicity
of congeners [3]. On this basis it can be suggested that PCBs do not
directly affect steroidogenesis in bovine ovary but they can impair the
stimulatory effect of LH and FSH on this process. Further, we showed
that PCB congeners increased OT secretion from granulosa cells, but
paradoxically each congener decreased FSH-stimulated OT secretion.
Likewise, congeners and Ar1248 stimulated OT secretion from luteal
cells, though this effect was dependent on the stage of the cycle and type
of congener, while the effect of PCBs on LH-stimulated OT secretion
was equivocal in course of the estrous cycle [12].
Mechanism of PCBs’ effect on the cell and within the cell is still
unclear. These pollutants are suggested to affect cell functioning via ER
and AhR. However, the effect of PCB 77 and 153 on OT secretion was
blocked by RU486 (blocker of glucocorticoid receptor; GR), but it did
not change the effect evoked by PCB126 in both granulosa and luteal
cells (unpublished data). These data suggest that PCBs can affect OT
secretion via both GR and ER. However, it was found in bovine granulosa
cells that the promoter region of the bovine NP/OT gene has only an
inactive form of response element for E2 [8], while it contains a response
element for orphan receptor SF-1 which can bind glucocorticoids [6].
Moreover, expression of SF-1 gene correlates well with expression of
the neurophysin/OT gene in the developing corpus luteum [17]. Hence,
it is difficult to state definitely whether GR or ER is predominantly
responsible for PCBs-stimulated OT secretion from a cell. Concluding, it
is suggested that bovine PCBs can indirectly impair ovarian functioning
via ovarian OT secretion/synthesis.
192
PCBs and phytoestrogens affect reproduction in cows
Effect of PCB on uterine functions
Myometrial strips were incubated for 24-72h with different doses of PCB
congeners at 4°C in aerated atmosphere. Thereafter contractility of strips
was registered by means of a special chamber of HSE Schuler Organbath
apparatus. PCBs significantly increased the spontaneous and OT-stimulated
frequency and force of contractions of longitudinal myometrial strips [20]. In
most cases this effect was time- and dose-dependent. Further, we found that
PCB congeners (10 ng/ml; 48h) increased basal intracellular concentrations
of Ca2+ [18]. This response of calcium mobilization challenged by OT was
significantly inhibited or delayed in cells incubated previously with PCB
congeners, compared to control. Moreover, we showed that all used PCBs
markedly stimulated PGF2α but not PGE2 secretion from endometrial cells,
hence the ratio of PGF2α:PGE2 was also increased [19]. Thus, the effect on
intracellular concentrations of Ca2+ in myometrial cells and on the ratio
of PGF2α:PGE2 secreted by endometrial cell could be a part of the cellular
mechanism by means of which PCBs increase myometrial contractility.
Involvement of phytoestrogens in PCBs’ diverse effect on ovarian and
uterine cells
Since PCBs and phytoestrogens can be bound by the same receptors
(e.g. ER), we have tested the hypothesis that phytoestrogens (genistein,
daidzein, coumestrol) can protect the cell against the described effect of
PCBs. It appeared that genistein inhibited the effect of all PCB congeners
on OT secretion in granulosa cells. Phytoestrogens increase OT secretion,
but when each of them is adminstered jointly with PCB congener, except
for PCB 126 with coumestrol, they decreased OT secretion from luteal
cells. Coumestrol itself reduced the force of myometrial contractions, and
coumestrol or daidzein inhibited PCBs-stimulated effect on myometrial
contractions (unpublished data). When endometrial cells were incubated
with each of the phytoestrogens, there was a decrease of both prostaglandins
secretion compared to the control, but without altering the PGF2α:PGE2
ratio. Moreover, phytoestrogens clearly reduced the amount of PGF2α
Kotwica et al.
193
elicited by all PCBs used in the study, and they can also reduce PGE2
secretion compared to that evoked by PCB 126 and 153 only [19]. Thus,
phytoestrogens can restore the proper ratio of PGF2α:PGE2 secreted by
bovine endometrium, if it was previously disturbed by PCBs.
In conclusion, these results suggest that PCBs and phytoestrogens
are involved in the regulation of ovarian steroidogenesis and uterine
contractility. But if PCBs disturb these processes, then phytoestrogens can
partly reduce or reverse their effects. Thus, a diet containing plant flavonoids
can be an important part of a feeding strategy to prevent reproduction
disorders in domestic animals elicited by environmental pollutants.
ACKNOWLEDGEMENTS
This study was supported by Solicited Project from KBN (PBZ-KBN-084/
P06/2002).
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