Leukotriene production profiles and actions in the bovine endometrium during the oestrous cycle.

We have previously shown the influence of leukotrienes (LTs) on reproductive functions in vivo: LTB4 is luteotrophic and supports corpus luteum function inducing PGE2 and progesterone (P4) secretion, whereas LTC4 is luteolytic and stimulates PGF2α secretion in cattle. The aim of this study was to examine expression and production profiles of LTs and their actions in the endometrium. LT receptors (LTB4R for LTB4 and CysLTR2 for LTC4), 5-lipoxygenase (LO), 12-LO synthase (LTCS) and LTA4 hydrolase (LTAH) mRNA and protein expression, as well as LT production were measured in bovine endometrial tissue during the luteal phases of the oestrous cycle. The action of LTs on uterine function was studied by measuring the level of PGs after stimulating uterine slices with LTs on Days 8-10 of the cycle. Expression of 5-LO and LTB4R mRNA and protein were highest on Days 2-4 of the cycle, while CysLTR2 and LTCS were highest on Days 16-18 (P<0.05). LTB4 concentration was highest on Days 2-4 of the cycle, whereas the greatest LTC4 level was on Days 16-18 (P<0.05). Both LTB4 and C4 increased the content of PGE2 and F2α in endometrial slices at a dose of 10(-7)M (P<0.05). In summary, mRNA expression and activation of receptors for LTB4 and production occur in the first part of the cycle, whereas LTC4 and its receptors predominate at the end of the cycle. The 12-LO and 5-LO pathways are complementary routes of LT production in the bovine uterus.

Regulation of prostaglandin biosynthesis by interleukin-1 in cultured bovine endometrial cells.

Interleukin-1 (IL1) has been shown to be a potent stimulator of prostaglandin (PG) production in bovine endometrium. The aim of the present study was to determine the cell types in the endometrium (epithelial or stromal cells) responsible for the secretion of PGE2 and PGF2alpha in response to IL1A, and the intracellular mechanisms of IL1A action. Cultured bovine epithelial and stromal cells were exposed to IL1A or IL1B (0.006-3.0 nM) for 24 h. IL1A and IL1B dose-dependently stimulated PGE2 and PGF2alpha production in the stromal cells, but not in the epithelial cells. The stimulatory effect of IL1A (0.06-3.0 nM) on PG production was greater than that of IL1B. The stimulatory actions of IL1A on PG production was augmented by supplementing arachidonic acid (AA). When the stromal cells were incubated with IL1A and inhibitors of phospholipase (PL) C or PLA2 (1 microM; anthranilic acid), only PLA2 inhibitor completely stopped the stimulatory action of IL1A on PG production. Moreover, a specific cyclooxygenase-2 (COX2) inhibitor blocked the stimulatory effect of IL1A on PG production. IL1A (0.06 nM) promoted COX2 and microsomal PGE synthase-1 (PGES1) gene and its protein expression. The expression of COX1, PGES2, PGES3, and PGF synthase (PGFS) mRNA was not affected by IL1A in the stromal cells. The overall results indicate that 1) the target of IL1A and IL1B for stimulating both PGE2 and PGF2alpha production is the stromal cells, 2) IL1A is a far more potent stimulator than IL1B on PG production in stromal cells, 3) the stimulatory effect of IL1A on PG production is mediated via the activation of PLA2 and COX2, and (4) IL1A induced PG production by increasing expressions of COX2 and PGES1 mRNAs and their proteins in bovine stromal cells.

Mechanism of phytoestrogens action in reproductive processes of mammals and birds.

Phytoestrogens are polyphenolic compounds that occur ubiquitously in food of plant origin and they have a variety of biological effects in numerous animal cell systems in vivo as well in vitro. Results of studies conducted on animals have shown that effects of phytoestrogens vary depending on species, sex, routes of administration, dose and exposure time. This review summarizes the results of ours studies concerning: 1/ molecular mechanism of phytoestrogen action in porcine granulosa cells, 2/ the involvement of phytoestrogens in immunological regulations of bovine corpus luteum function during luteolysis, 3/ genistein action on metabotropic hormones and lipid-carbohydrate metabolism in rats during pregnancy, 4/ the effects of phytoestrogens on reproductive processes in males of bilgoraj goose.

Phyto- and endogenous estrogens differently activate intracellular calcium ion mobilization in bovine endometrial cells.

The main purpose of this study was to check whether phyto- and endogenous estrogens influence calcium ion mobilization [Ca(2+)](i) in bovine endometrial cells and whether this action is connected with biological effects i.e. prostaglandin (PG)F(2alpha) production. In our study we used two calcium measurement methods by comparing the microscopic method with widely used quantitative - spectrofluorometric method of [Ca(2+)](i) measurement. We also wanted to confirm whether visualization of calcium ion [Ca(2+)](i) in cells using microscopic method supported by micro image analysis (Micro Image Olympus system) reflects real, qualitative changes in the ion concentration. In both methods a cell-permeable form of fluorescent [Ca(2+)](i) indicator Fura-2 was used. Cultured bovine endometrial epithelial and stromal cells influenced by phorbol-2-myristate-13-acetate (PMA; positive control), estradiol 17-beta (E(2); endogenous estrogen) and active metabolites of phytoestrogens (environmental estrogens) were used as a model to study PGF(2alpha) secretion and [Ca(2+)](i) mobilization in the cells. Equol and para-ethyl-phenol in doses of 10(-8)-10(-6) M increased PGF(2alpha) concentration both in epithelial and stromal cells (P<0.05). In both methods, equol and para-ethyl-phenol did not cause intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P>0.05). Both methods revealed that only E(2) and PMA induced intracellular [Ca(2+)](i) mobilization in epithelial and stromal cells (P<0.05). The results of both methods were highly correlated (P<0.001; r=0.82 for epithelial cells and r=0.89 for stromal cells). In conclusion, both methods gave approximately the same results and showed that phytoestrogens, in contrast to PMA and E(2), did not cause intracellular [Ca(2+)](i) mobilization in endometrial cells. The obtained results proved that the [Ca(2+)](i) visualization method supported by micro image analysis can produce similar results to the spectrofluorometric method.

Equol and para-ethyl-phenol stimulate prostaglandin F(2alpha) secretion in bovine corpus luteum: intracellular mechanisms of action.

Corpus luteum (CL) is a reproductive gland that plays a crucial endocrine role in the regulation of the estrous cycle, fertility, and pregnancy in cattle. The main function of CL is secretion of progesterone (P4), an important hormone for establishment a successful pregnancy, whereas prostaglandin F(2alpha) (PGF(2alpha)), 17beta-estradiol (E(2)) and testosterone (T) are implicated in the regulation of luteolysis. It has been shown that phytoestrogens may disrupt numerous reproductive functions on several levels of regulation and via different intracellular mechanisms. Using a cell-culture system of steroidogenic cells of the bovine CL, we determined effects of active phytoestrogen metabolites (equol and para-ethyl-phenol) on PGF(2alpha), P4, and T synthesis in steroidogenic CL cells. Moreover, we examined the intracellular mechanisms of phytoestrogen metabolite actions. Phytoestrogen metabolites did not affect P4 production in steroidogenic CL cells. However, PGF(2alpha) and T were significantly stimulated by metabolites of phytoestrogens in the bovine steroidogenic CL cells. To study the intracellular mechanism of endogenous E(2) and phytoestrogen metabolites action, steroidogenic cells were preincubated with a phospholipase C inhibitor (U73122), a protein kinase C inhibitor (staurosporine), an estrogen receptor antagonist (ICI) and a transcription inhibitor (actinomycin D) for 0.5h, and then stimulated with para-ethyl-phenol, equol or E(2). Only U73122 and staurosporine totally reduced the stimulatory effect of E(2) on PGF(2alpha) production by the cells. ICI and actinomycin D only partially reduced E(2) action on CL cells. In contrast, the stimulatory effect of phytoestrogen metabolites was totally inhibited by ICI and actinomycin D. Moreover, in contrast to E(2) action, phytoestrogen metabolites did not cause intracellular calcium mobilization in the cells. The present study demonstrated that phytoestrogen metabolites stimulate PGF(2alpha) secretion in steroidogenic cells of the bovine CL via the estrogen receptor-dependent, genomic pathway.

Phytoestrogens and their metabolites inhibit the sensitivity of the bovine corpus luteum to luteotropic factors.

The aim of this study was to examine whether active metabolites of phytoestrogens (equol and para-ethyl-phenol) inhibit sensitivity of bovine corpus luteum (CL) to luteinizing hormone (LH) and to auto/paracrine luteotropic factors (prostaglandin E2-PGE2 and prostaglandin F(2alpha)-PGF(2alpha)), and whether they influence pulsatile progesterone (P4) secretion by the bovine CL. In in vivo experiments, high levels of equol and para-ethyl-phenol were found in plasma and in the CL tissue of heifers and cows fed a soy bean diet (2.5 kg/animal/day), along with lower concentrations of P4 (P < 0.05). Both Prostaglandins (PG) and LH strongly stimulated P4 secretion in cultured pieces of CL that were collected from cows fed a standard diet (P < 0.01). There was no effect of PGs and LH on P4 stimulation in CLs obtained from cows fed a diet rich in soy bean. Finally, we examined whether active metabolites of phytoestrogens participated in regulation of pulsatile P4 secretion and LH-stimulated P4 secretion in vitro using a microdialysis system. Equol and para-ethyl-phenol had no effect on basic and pulsatile P4 secretion in CLs during 240 min of perfusion when compared to the control (P < 0.05). However, they inhibited LH-stimulated P4 secretion (P < 0.05). Phytoestrogens and their metabolites may disrupt CL function by inhibiting PG- and LH-stimulated P4 secretion.

Phytoestrogens modulate prostaglandin production in bovine endometrium: cell type specificity and intracellular mechanisms.

Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2alpha, whereas PGE2 has luteotropic actions. Estradiol 17beta (E2) regulates uterus function by influencing PG synthesis. Phytoestrogens structurally resemble E2 and possess estrogenic activity; therefore, they may mimic the effects of E2 on PG synthesis and influence the reproductive system. Using a cell-culture system of bovine epithelial and stromal cells, we determined cell-specific effects of phytoestrogens (i.e., daidzein, genistein), their metabolites (i.e., equol and para-ethyl-phenol, respectively), and E2 on PGF2alpha and PGE2 synthesis and examined the intracellular mechanisms of their actions. Both PGs produced by stromal and epithelial cells were significantly stimulated by phytoestrogens and their metabolites. However, PGF2alpha synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2alpha than stromal cells, increasing the PGF2alpha to PGE2 ratio. The epithelial and stromal cells were preincubated with an estrogen-receptor (ER) antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) for 0.5 hrs and then stimulated with equol, para-ethyl-phenol, or E2. Although the action of E2 on PGF2alpha synthesis was blocked by all reagents, the stimulatory effect of phytoestrogens was blocked only by ICI and actinomycin D in both cell types. Moreover, in contrast to E2 action, phytoestrogens did not cause intracellular calcium mobilization in either epithelial or stromal cells. Phytoestrogens stimulate both PGF2alpha and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2alpha synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2alpha to PGE2 ratio.

Soybean-derived phytoestrogens regulate prostaglandin secretion in endometrium during cattle estrous cycle and early pregnancy.

Phytoestrogens acting as endocrine disruptors may induce various pathologies in the female reproductive tract. The purpose of this study was to determine whether phytoestrogens present in the soybean and/or their metabolites are detectable in the plasma of cows fed a diet rich in soy and whether these phytoestrogens influence reproductive efficiency and prostaglandin (PG) synthesis during the estrous cycle and early pregnancy in the bovine endometrium. In in vivo Experiment 1, we found significant levels of daidzein and genistein in the fodder and their metabolites (equol and p-ethyl-phenol) in bovine serum and urine. The mean number of artificial inseminations (AIs) and pregnancy rates in two kinds of herds, control and experimental (cows fed with soybean 2.5 kg/day), were almost double in the soy-diet herd in comparison with the control animals. In in vivo Experiment 2, three out of five heifers fed soybean (2.5 kg/day) became pregnant whereas four out of five heifers in the control group became pregnant. The concentrations of a metabolite of PGF2alpha (PGFM) were significantly higher in the blood plasma of heifers fed a diet rich in soybean than those in the control heifers throughout the first 21 days after ovulation and AI. The higher levels of PGFM were positively correlated with equol and p-ethyl phenol concentrations in the blood. In in vitro experiments, the influence of isoflavones on PG secretion in different stages of the estrous cycle was studied. Although all phytoestrogens augmented the output of both PGs throughout the estrous cycle, equol and p-ethyl-phenol preferentially stimulated PGF2alpha output. The results obtained lead to the conclusion that soy-derived phytoestrogens and their metabolites disrupt reproductive efficiency and uterus function by modulating the ratio of PGF2alpha to PGE2, which leads to high, nonphysiological production of luteolytic PGF2alpha in cattle during the estrous cycle and early pregnancy.