Chloroorganic (DDT) and organophosphate (malathion) insecticides impair the motor function of the bovine cervix.

Dichlorodiphenyltrichloroethane (DDT) is a representative organochlorine insecticide and a known endocrine disruptor. Malathion is an organophosphate insecticide and a next-generation pesticide. Previously, it was shown that oxytocin (OT) and prostaglandins (PGs) are involved in the mechanism of the adverse effect of DDT on bovine myometrial contractions. However, disruption of myometrial contractions without disruption of cervical activity may not be sufficient to cause preterm delivery. Hence, the aim of this study was to determine the effects of insecticides on the function of the bovine cervix at preovulation period. Bovine cervical cells or strips were treated with DDT or malathion (0.1-100 ng/ml), and neither DDT nor malathion (each at a dose of 100 ng/ml) affected the viability of cervical cells. Malathion (0.1-10 ng/ml) and the high doses of DDT (10 ng/ml) decreased the force of cervical contractions, in contrast to a low dose of DDT (0.1 ng/ml). Both insecticides also decreased the mRNA expression of the OT receptor and the level of the second messenger (inositol triphosphate, IP3). Moreover, DDT decreased the amount of other second messengers (diacylglycerol, DAG), while malathion decreased the amount of gap junction protein (GAP). Only malathion increased PGE2 and decreased PGF2α secretion, while neither insecticide had an effect on both prostaglandins synthesis. Both DDT and malathion impaired cervical contractions, secretory function and cellular signalling. It is also possible that malathion-mediated induction of locally produced PGE2 can be followed by cervical softening. Admittedly it was shown that DDT and malathion can evoke failures in the regulation of motor function of cervix during oestrus cycle, while their harmful effect on gestation can be also not excluded.

The effect of DDE and dieldrin and their parental substances (DDT and aldrin) on PGE2 and PGF2α release from bovine endometrial explants collected during 120 to 180 days of the gestational period.

Dieldrin and DDE are environmental metabolites of the organochlorine pesticides aldrin and DDT, respectively. During pregnancy, these chemicals can quickly infiltrate through the placental barrier, accumulate in amniotic fluid and fetus, and act as endocrine disruptors (EDs). The aim of this study was to investigate the effect of DDE and dieldrin and their parental substances at concentrations of 1 and 10 ng/ml on secretion of PGE2 and PGF2α from bovine endometrial explants (120-150 and 151-180 days of pregnancy) after 24 hr of incubation with EDs. The mRNA expression of COX2, PGES and PGFS and the concentrations of PGE2 and PGF2α were measured. EDs did not affect (p>0.05) COX2 gene expression, but DDT and DDE decreased (p⟨0.05) PGES expression and PGE2 secretion in the explants from 120-150 days of pregnancy. Depending on the dose, DDT and DDE increased (p⟨0.05) PGFS expression and PGF2α secretion from the explants from 120-150 days and decreased PGF2α secretion (p⟨0.05) from the explants from 151-180 days of pregnancy. Aldrin and dieldrin decreased (p⟨0.05) PGFS expression and PGF2α secretion from all explants. In summary, EDs disrupt the secretion of PGE2 and PGF2α by influencing the gene expression of PGES and PGFS.

Effects of DDT, DDE, aldrin and dieldrin on prostaglandin, oxytocin and steroid hormone release from smooth chorion explants of cattle.

Chlorooganic xenobiotics (XBs) such as DDT, DDE, aldrin and dieldrin interfere with release of hormones from chorionic villi that are necessary for sustaining the normal course pregnancy: prostaglandins (PGs), oxytocin (OT), progesterone (P4) and estradiol (E2). Approximately 20 %-40 % of these hormones originate from the smooth chorion. The aim of current studies was to investigate effects of these XBs on synthesis and release of PGE2, PGF2α, OT, P4 and E2 from explants of smooth chorion of cattle, obtained during the120-150 and 151-180 day gestational period. Explants were incubated with DDT, DDE, aldrin or dieldrin at concentrations of 1 and 10 ng/mL for 24 h, and concentrations of PGE2, PGF2α, OT, P4 and E2 in post incubation medium and the relative abundances of COX-2, PTGES, AKR1B1, NP-I/OT, PAM, HSD3B, and CYP19A1 mRNA transcripts in tissue explants were determined. The XBs did not have effects on cell viability in explants (P > 0.05), however, there were effects on prostaglandins, OT and P4 secretion and relative abundance of mRNA transcript for genes encoding the main enzymes involved in synthesis of these hormones (P < 0.05). The XBs that were evaluated did not have effects on E2 synthesis and secretion (P > 0.05). In summary, XBs evaluated in the present study had effects on the pattern of prostaglandin secretion, and can increase OT and P4 release from smooth chorion explants. Because XBs inhibit hormonal action throughout the chorion, there is an increase in risk of abortions or premature births in animals.

The orphan nuclear receptor SF-1 is involved in the effect of PCBs, DDT, and DDE on the secretion of steroid hormones and oxytocin from bovine luteal cells during the estrous cycle in vitro.

The orphan receptor steroidogenic factor-1 (SF-1) is involved in the regulation of ovarian steroidogenesis in cows. It is hypothesized that estrogen-like chlorinated compounds might affect SF-1, and thus impair the function of the ovary. Bovine luteal cells from the estrous cycle (Days: 1-5, 6-10, 11-15, and 16-19) were treated for 50 hours with DDT, 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene, 3,3'4,4'-tetrachlorobiphenyl or 2'2'4,4',5,5'-hexachlorobiphenyl (each at a dose of 10 ng/mL). Luteal cells were also treated with 4-(heptyloxy)phenol (1 × 10(-7) M), an SF-1 agonist, and F0160 (1 × 10(-6) M), an SF-1 blocker, jointly or separately. The secretion of progesterone and oxytocin and the expression of oxytocin precursor (NP-I/OT) messenger RNA were increased (P < 0.05) by all studied xenobiotics and 4-(heptyloxy)phenol, although they were inhibited (P < 0.05) by F0160. However, the xenobiotics did not affect (P > 0.05) SF-1 messenger RNA expression. In summary, SF-1 is involved in the adverse effect of chlorinated xenobiotics on the regulation of the bovine CL.

Involvement of the orphan nuclear receptor SF-1 in the effect of PCBs, DDT and DDE on the secretion of steroid hormones and oxytocin from bovine granulosa cells.

Polychlorinated biphenyls (PCBs), DDT and its metabolite (DDE) belong to estrogen-like endocrine disruptors. However, though their activity is approximately 1000-fold lower than the activity of estradiol (E2), this steroid's high concentration in follicular fluid and incubation media does not inhibit the influence of these xenobiotics. It was hypothesized that these xenobiotics might affect Steroidogenic Factor-1 (SF-1) and impair ovary function. To test this hypothesis, granulosa cells were obtained from ovarian follicles >1 or <1cm in diameter, which were treated with PCB-77, PCB-153, DDT or DDE (each at 10ng/ml), alone or jointly with an SF-1 antagonist (F0160). Treatment with the SF-1 antagonist inhibited (P<0.05) the secretion of P4 from cells of both sizes of follicles, as induced (P<0.05) by an SF-1 activator (HxP), DDE or PCB-153. All xenobiotics and HxP stimulated (P<0.05) the synthesis and secretion of oxytocin (OT). However, the effect on mRNA expression for NP-I/OT, which is OT precursor, was inhibited (P<0.05) by F0160 in all cultures treated with PCB-77, except for granulosa cells derived from follicles <1cm. Moreover, F0160 inhibited the effect on OT secretion of HxP, as well as all xenobiotics except for PCB-77 and DDE, in granulosa cells derived from follicles <1cm. Xenobiotic treatment did not affect (P>0.05) the expression for SF-1 mRNA. It is suggested that the SF-1 receptor may be involved in the adverse effects of xenobiotics on P4 secretion as well as the synthesis and secretion of OT.

The expression of Steroidogenic Factor-1 and its role in bovine steroidogenic ovarian cells during the estrus cycle and first trimester of pregnancy.

The orphan receptor Steroidogenic Factor-1 (SF-1, NR5A1), a member of the nuclear receptor superfamily, is present in fetal and adult steroidogenic tissues and also participates in the regulation of ovarian function. In this study, the expression levels of SF-1 mRNA and protein were determined in granulosa cells (from follicles >1cm and <1cm in diameter) and luteal tissue (from days 1-5, 6-10, 11-15, and 16-19 of the estrous cycle and weeks 3-5, 6-8, and 9-12 of pregnancy). Additionally, the effects of a synthetic SF-1 stimulator (4-(heptyloxy)phenol - HxP; 1×10(-7)M) and a synthetic SF-1 inhibitor (F0160; 1×10(-5)M) on the secretion of estradiol and oxytocin (OT) from granulosa cells (from follicles>1cm) and the secretion of progesterone (P4) and OT from luteal cells (days 11-16 of the estrous cycle) were investigated. The levels of SF-1 mRNA and protein were higher in granulosa cells (P<0.05) from follicles>1cm than in cells from follicles<1cm. In luteal tissue, the mRNA abundance was the highest (P<0.05) on days 6-10 of the estrous cycle, and the amount of protein was the highest on days 6-15 (P<0.05). The lowest levels of mRNA and protein for SF-1 were observed on days 16-19 of the estrous cycle (P<0.05). The abundance of SF-1 mRNA decreased at 9-12 weeks of pregnancy (P<0.05). The stimulation of the studied cells with HxP increased P4 and estradiol secretion from luteal and granulosa cells, respectively, and OT secretion from both types of cells. The SF-1 inhibitor did not affect hormone secretion by either type of cell, but it did diminish the effect induced by the SF-1 stimulator. The obtained data revealed estrous cycle-dependent levels of mRNA and protein for SF-1 in luteal tissue, and the use of a specific SF-1 stimulator and a specific SF-1 inhibitor confirmed the involvement of this receptor in steroidogenesis and OT secretion from cultured granulosa and luteal cells. These findings suggest that the SF-1 receptor participates in the local regulation of ovarian function during both the estrous cycle and the first trimester of pregnancy in cows. Furthermore, the concentrations of the SF-1 inhibitor and stimulator that we used in the primary cell culture could effectively modify the activity of this receptor.

Adverse influence of coumestrol on secretory function of bovine luteal cells in the first trimester of pregnancy.

Coumestrol is one of a few biologically active substances present in leguminous plants, which are widely used as fodder for ruminants. Depending on the doses, coumestrol acts on the reproductive processes as an estrogen-like factor or antiestrogen to evoke a decrease in ovulation frequency, elongation of estrous cycle duration. The aim of the current investigations was to study the influence of coumestrol on secretory function of luteal cells obtained from first trimester of pregnant cows. Luteal cells (2.5 × 10(5) /mL) from 3rd to 5th, 6th to 8th, and 9th to 12th week of pregnancy were preincubated for 24 h and incubated with coumestrol (1 × 10(-6) M) for successive 48 h and the medium concentrations of progesterone (P4), oxytocin (OT), prostaglandin (PG) E2 and F2α were determined. Moreover, the expression of mRNA for neurophysin-I/oxytocin (NP-I/OT; precursor of OT) and peptidyl-glycine-α-amidating mono-oxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. Coumestrol did not affect P4 secretion but increased the secretion of OT from the cells collected at all stages of gestation studied. Hence, the ratio of P4 to OT was markedly decreased. Simultaneously, coumestrol increased the expression of NP-I/OT mRNA during 9th to 12th weeks of pregnancy, and mRNA for PGA during 3rd to 5th and 9th to 12th weeks of gestation. Furthermore, coumestrol decreased PGE2 secretion from luteal cells in all studied stages of pregnancy, while it affected PGF2α metabolite (PGFM) concentration only from week 3 to 5 of pregnancy. Obtained results suggest that coumestrol impairs secretory function of the corpus luteum (CL) and this way it can affect the maintenance of pregnancy in the cow.

The adverse effect of phytoestrogens on the synthesis and secretion of ovarian oxytocin in cattle.

The current investigations were undertaken to study the mechanism of the adverse effect of phytoestrogens on the function of bovine granulosa (follicles >1< cm in diameter) and luteal cells from day 1-5, 6-10, 11-15, 16-19 of the oestrous cycle. The cells were incubated with genistein, daidzein or coumestrol (each at the dose of 1 × 10(-6) m). The viability and secretion of estradiol (E2), progesterone (P4) and oxytocin (OT) were measured after 72 h of incubation. Moreover, the expression of mRNA for neurophysin-I/OT (NP-I/OT; precursor of OT) and peptidyl-glycine-α-amidating monooxygenase (PGA, an enzyme responsible for post-translational OT synthesis) was determined after 8 h of treatment. None of the phytoestrogens used affected the viability of cells except for coumestrol. The increased secretion of E2 and P4 was only obtained by coumestrol (p<0.05) from granulosa cells from follicles <1cm in diameter and decreased from luteal cells on days 11-15 of the oestrous cycle, respectively. All three phytoestrogens stimulated (p<0.05) OT secretion from granulosa and luteal cells in all stages of the oestrous cycle and the expression of NP-I/OT mRNA in the both types of cells. The expression of mRNA for PGA was stimulated (p<0.05) by daidzein and coumestrol in granulosa cells, and by genistein and coumestrol in luteal cells. In conclusion, our results demonstrate that these phytoestrogens can impair the ovary function in cattle by adversely affecting the synthesis of OT in follicles and in corpus luteum. However, their influence on the ovarian steroids secretion was less evident.