Michal H. Wrobel

Effect of environmental pollutants on oxytocin synthesis and secretion from corpus luteum and on contractions of uterus from pregnant cows

Chloro-organic compounds are persistent environmental pollutants and affect many reproductive processes. Oxytocin (OT) synthesized in luteal cells is a local regulator of ovarian activity and uterine contractions. Therefore the effect of xenobiotics on the OT prohormone synthesis, secretion of OT and progesterone (P4) from luteal cells and on myometrial contractions during early pregnancy in cows was investigated. Luteal cells and myometrial strips from a cow at early pregnancy were treated with polychlorinated biphenyl 77 (PCB 77), dichlorodiphenyltrichloroethane (DDT), dichlorodiphenyldichloroethylene (DDE) and hexachlorocyclohexane (HCH) (1 or 10 ng/ml). The mRNA expression of neurophysin-I/oxytocin (NP-I/OT) and peptidyl-glycine-alpha-amidating mono-oxygenase (PGA) and concentration of OT and P4 were determined by RT-PCR and EIA, respectively. Moreover, the effect of xenobiotics given with P4 (12 ng/ml) on the basal and OT (10(-7)M) stimulated contractions of myometrial strips was studied. Xenobiotics increased (P<0.05) OT secretion but DDE only stimulated P4 secretion. The ratio of P4 to OT in culture medium was decreased by all xenobiotics during 9-12 weeks of pregnancy. All xenobiotics, except HCH, increased (P<0.05) mRNA expression of NP-I/OT during all stages of pregnancy and all treatments decreased (P<0.05) expression of mRNA for PGA during 9-12 weeks of pregnancy. Myometrial strips were relaxed (P<0.01) after pre-incubation with P4, while each of the xenobiotics jointly with P4 increased (P<0.01) myometrial contractions. In conclusion, the xenobiotics used increased both expression of mRNA for genes involved in OT synthesis and secretion of OT from luteal cells. This decreases the ratio of P4 to OT and presumably, in this manner, the chloro-organic compounds can influence uterine contractions and enhance risk of abortions in pregnant females.

Involvement of prostaglandin F2α in the adverse effect of PCB 77 on the force of contractions of bovine myometrium

Polychlorinated biphenyls (PCBs) stimulate in vitro both the force of myometrial contractions and endometrial secretion of PGF2alpha in cattle. Therefore, the goal of this study was to investigate the participation of PGF2alpha in the effect of PCBs on uterine contractility. For this aim, the myometrial strips were incubated (48h) with PCB 77 at the dose of 1, 10 and 100ng/ml (i.e., 0.0034, 0.034 and 0.34nmol/ml) separately or jointly with indomethacin (INDO, 10(-4)M), which blocks the PGF2alpha synthesis. Next, the force of myometrial strips contractions was measured. Further, the influence of PCB 77 (0.1, 1 and 10ng/ml) on the PGF2alpha secretion from myometrial cells after 6, 24, and 48h and PCB 77 (1 and 10ng/ml) on the mRNA expression of cyclooxygenase 2 (COX-2) and PGF2alpha synthase (PGFS) in myometrial cells after 6 and 24h, was investigated. The increase (P<0.05-0.001) of the contractions force of myometrial strips evoked by each dose of PCB 77, was markedly reduced (P<0.05-001) by INDO. There was an increase (P<0.05-0.001) of both PGF2alpha secretion after all studied periods of cell incubation and mRNA expression for COX-2 and PGFS after 6h treatment of myometrial cells with PCB 77. It can be concluded that myometrial synthesis of PGF2alpha and its further secretion is a part of the mechanism by means of which PCB 77 may affect the force of myometrial contractions in cattle.

The adverse effects of aldrin and dieldrin on both myometrial contractions and the secretory functions of bovine ovaries and uterus in vitro

Aldrin and dieldrin are chloroorganic insecticides which are recognised as endocrine disruptors. The aim of the study was to investigate their effect on the secretory functions of the uterus and ovary and on myometrial contractions. Myometrial strips and uterine and ovarian cells from nonpregnant cows were incubated with the xenobiotics (0.1, 1 or 10 ng/ml) for 24 or 72 h. Next, their effect on viability of myometrial, endometrial, granulosa and luteal cells, myometrial strip contractions, the synthesis and secretion of prostaglandins (PGs: PGF2α and PGE2) from uterine cells, the secretion of oestradiol (E2), testosterone (T) and oxytocin (OT) from granulosa cells and the secretion of progesterone (P4) and OT from luteal cells were determined. Neither of the xenobiotics (10 ng/ml) affected (P>0.05) the viability of the ovarian and uterine cells, while both (0.1-10 ng/ml) decreased (P<0.05) the basal and OT-stimulated myometrial contractions. In spite of these effects, neither of the insecticides affected (P>0.05) the synthesis and the secretion of PGs from the myometrial cells. Although they also did not impair the secretion of the PGs from the endometrial cells, they abolished (P<0.05) the stimulatory effect of OT (P<0.05) on the secretion of the PGs and stimulated (P<0.05) the secretion of OT from the granulosa and luteal cells. Moreover, aldrin and dieldrin stimulated secretion of E2 and T from the granulosa cells, while only dieldrin increased (P<0.05) the secretion of P4 from luteal cells. The data show that aldrin and dieldrin stimulated the secretory function of the cultured granulosa and luteal cells and inhibited the myometrial contractions of cows in vitro, which may affect on natural parturition.

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.

Impairment of uterine smooth muscle contractions and prostaglandin secretion from cattle myometrium and corpus luteum in vitro is influenced by DDT, DDE and HCH

The aim of this study was to investigate the effect of dichlorodiphenyltrichloroethane(DDT), dichlorodiphenyldichloroethylene (DDE) and γ-hexachlorocyclohexane (HCH) (10 ng/ml) on myometrial motility and the secretory function of the myometrium and corpus luteum (CL) collected from cows on days 8-12 of the estrous cycle. All of the xenobiotics increased (P<0.05) myometrial contractility. Moreover, the xenobiotics stimulated the secretion of the following prostaglandins (PGs) from myometrial strips: PGF2α, PGE2 and PGI2. DDT and DDE also increased (P<0.05) the release of PGF2α from CL strips, and HCH had the same effect (P<0.05) on the secretion of PGE2 and PGI2. The studied xenobiotics did not affect (P>0.05) PG synthesis, but DDT and DDE increased the mRNA expression levels of leukemia inhibitor factor (LIF), which can stimulate PG production. In summary, the xenobiotics affected PG secretion from cow myometrium and CL, which may contribute to the mechanism of uterine contraction disturbance.

Influence of polychlorinated biphenyls and their hydroxylated metabolites on prostaglandins secretion from epithelial cells of bovine oviduct, in vitro.

Polychlorinated biphenyls (PCBs) markedly stimulate bovine uterine contractions and prostaglandin (PG) F2alpha secreted from both, myometrial and endometrial cells is essentially involved in this process. Since contractions of the oviduct are crucial for gametes and embryo transport, therefore the goal of this study was to investigate the influence of PCBs on PGF2alpha and PGE2 secretion from oviductal epithelium. Epithelial cells of the oviduct, from cows and heifers on days 1-5 of estrous cycle, were treated with PCBs: technical mixture (Aroclor 1248; Ar 1248), individual congeners (PCB 30 and PCB 153) and hydroxylated metabolites (PCB 30-OH and PCB 50-OH). Viability of the cells after treatment with PCBs (10 and 100 ng/ml) was determined after 24, 48 and 72 h. The concentration of PGFM (metabolite of PGF2alpha) and PGE2 in culture medium was determined after 2 and 48 h of incubation with PCBs (0.1, 1 and 10 ng/ml). None of the PCBs affected (P>0.05) cell viability, whereas all of them, except PCB 30 after 48 h of treatment, increased (P<0.05-0.01) PGF2alpha secretion from epithelial cells. All PCBs also stimulated (P<0.05) the PGE2 secretion after 2h of incubation, but this effect was less evident or there was no effect after 48 h of treatment. We conclude that oviductal secretion of PGF2alpha and PGE2 is affected by PCBs and this can be a part of the mechanism by means of which PCBs may affect the contractions of bovine oviduct.

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.

The effect of DDT and its metabolite (DDE) on prostaglandin secretion from epithelial cells and on contractions of the smooth muscle of the bovine oviduct in vitro

The insecticide DDT and its metabolite (DDE), due to their lipolytic nature and resistance to biodegradation, are accumulated in the living tissues. In cows, DDT and DDE were found to affect prostaglandin (PG) secretion from the endometrium and contractions of the myometrium. In this study, the impact of both xenobiotics (0.1, 1, 10 or 100ng/ml) on the function of epithelial cells and muscle strips of bovine oviducts from 1 to 5day of the oestrous cycle was examined. Therefore the concentration of PGE2 and PGFM (a metabolite of PGF2α) in culture media, mRNA expression of genes involved in PGs synthesis in epithelial cells and the force and amplitude of strips contractions were measured after 2 and 24 or 48h of incubation. Neither DDT nor DDE affected the viability of cells after 48h (P>0.05). Both DDT and DDE increased the concentrations of PGFM in culture medium and secretion of PGE2 after only 2h of cell culture (P<0.05). Similar effects were seen for the influence of DDE on amount of PGFM after 48h, while DDT decreased secretion of PGE2 (P<0.05). DDT after 2h increased (P<0.05) mRNA expression of PGF2α synthase (PGFS), while both xenobiotics decreased (P<0.05) mRNA expression of cyclooxygenase-2 (COX-2) after 24h. DTT also increased the force of isthmus contractions after 2h, as did both xenobiotics after 48h (P<0.05). Moreover, after 2 and 48h, DDE stimulated the amplitude of contractions of the isthmus as well as the ampulla, (P<0.05). The effect of both compounds on oviduct contractions was diminished by indomethacin, which blocks PG synthesis. We conclude that oviductal secretion of prostaglandins is affected, by DDT and DDE. The influence of these xenobiotics on PGF2α and PGE2 secretion and ratio may be part of the mechanism by which both DDT and its metabolite disturb the contractions of oviductal muscle.

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 × 105/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.

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 steroids 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.