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Experimental Biology and Medicine 230:326-333 (2005)
© 2005 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Phytoestrogens Modulate Prostaglandin Production in Bovine Endometrium: Cell Type Specificity and Intracellular Mechanisms

Izabela Woclawek-Potocka*, Tomas J. Acosta{dagger}, Anna Korzekwa*, Mamadou M. Bah*, Masami Shibaya{dagger}, Kiyoshi Okuda{dagger} and Dariusz J. Skarzynski*,1

* Department of Reproductive Immunology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn, Poland; and {dagger} Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan

1To whom requests for reprints should be addressed at Laboratory of Reproductive Endocrinology, Faculty of Agriculture, Okayama University, Okayama 700-8530, Japan. E-mail: skadar{at}pan.olsztyn.pl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Prostaglandins (PGs) are known to modulate the proper cyclicity of bovine reproductive organs. The main luteolytic agent in ruminants is PGF2{alpha}, whereas PGE2 has luteotropic actions. Estradiol 17ß (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 PGF2{alpha} 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, PGF2{alpha} synthesis by both kinds of cells was greater stimulated than PGE2 synthesis. Moreover, epithelial cells treated with phytoestrogens synthesized more PGF2{alpha} than stromal cells, increasing the PGF2{alpha} 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 PGF2{alpha} 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 PGF2{alpha} and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2{alpha} synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2{alpha} to PGE2 ratio.

Key Words: cattle • endometrium • phytoestrogens • PGF2{alpha} • PGE2


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Soy phytoestrogens have been the subject of many reviews that describe their potential health benefits for both humans and animals (1–3). On the other hand, these substances also have some hazardous effects, especially on animals fed pasture that is rich in phytoestrogens (4). Daidzein and genistein are two major phytoestrogens that are present in soy (5, 6). In ruminants, rumen micro-organisms convert daidzein into equol and genistein into para-ethyl-phenol (7). There is increasing evidence that phytoestrogens can disrupt the reproductive processes in various species including humans (8, 9), rats (10), and cows (11). Phytoestrogens are also shown to inhibit the secretion of hypophyseal luteinizing hormone in the rat (12). Low levels of luteinizing hormone cause a decrease of progesterone (P4) production which, in turn, leads to high abortion rate (13). The decrease of pregnancy rate can also be attributed to phytoestrogen-dependent inhibition of the production of endogenous estrogens, which leads to disturbances in follicle development (9, 14). Thus, phytoestrogens acting as antagonists or/and agonists of endogenous estrogens may disrupt numerous reproductive processes on several levels of regulation.

In ruminants, endogenous estrogens are known to control the length of the estrous cycle by influencing prostaglandin (PG) synthesis (see Ref. 15 for a review). For example, the removal of estrogens on Day 8 of the cycle by destroying ovarian follicles with X-irradiation in ewes resulted in prolongation of the estrous cycle and lack of luteolysis (16). On the other hand, administration of estradiol 17ß (E2) to heifers on Day 13 of the cycle initiated luteolysis by increasing the PGF2{alpha} concentration (17). We have recently shown that soy bean–derived phytoestrogens regulate both PGF2{alpha} and PGE2 secretion in vivo in endometrium during the estrous cycle and early pregnancy in cattle (18, 19). In ruminants, PGF2{alpha} is the major luteolytic agent (20), whereas PGE2 has luteoprotective and anti-luteolytic properties (21, 22). Therefore, achieving an optimal PGF2{alpha} to PGE2 ratio is essential for endometrial receptivity, maintenance of corpus luteum (CL), and P4 secretion (23). It has been demonstrated that endometrial epithelial cells synthesize mainly PGF2{alpha}, whereas endometrial stromal cells synthesize approximately 10 times more of luteotropic PGE2 than epithelial cells (22, 24, 25). Therefore, it is important to determine which cells are target cells for their action of crude phytoestrogens and their metabolites in bovine endometrium.

Phytoestrogens have structural similarity to E2. Therefore, we suppose that they elicit or selectively modulate genomic estrogenic responses by binding to both {alpha} and ß estrogen receptors (ERs) (26, 27). We also suppose that phytoestrogens elicit or selectively modulate nongenomic estrogenic responses by their influence on protein kinase A action and intracellular calcium (Ca2+) mobilization (28–30).

The aims of the present study were to determine (i) which endometrial cells, epithelial or stromal, are the target of phytoestrogens and their metabolites for PG synthesis, (ii) what is the intracellular mechanism of the phytoestrogen-dependent increase of PGF2{alpha} synthesis in endometrial epithelial and stromal cells, and (iii) whether phytoestrogens mobilization in endometrial and E2 cause intracellular Ca2+ epithelial and stromal cells. In the present study, we selected two major soy-derived phytoestrogens (i.e., daidzein, genistein) and their metabolites (i.e., equol and para-ethyl-phenol, respectively), which have been identified in the serum of cows fed diets rich in soy bean (18, 19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Collection of Endometrial Tissue.
Bovine uteri were obtained at a local abattoir within 30 mins of exsanguination and were transported, on ice, to the laboratory within 1 hr. Estimation of the stages of the estrous cycle was determined by macroscopic observation of the ovaries and uteri (31). The uterine horns were separated from each other and from the remaining tissue.

Cell Isolation, Culture, and Experiments.
In this study, uteri of the early estrous cycle (i.e., Days 2–5) were used. The epithelial and stromal cells from the bovine endometrium were enzymatically separated (0.05% collagenase; #C0130; Sigma-Aldrich, St. Louis, MO) using procedures previously described (25). Cell viability was higher than 85%. The obtained cells consisted of stromal and epithelial cells with only a few fibroblasts.

The final pellet of both the stromal and epithelial cells was suspended in a culture medium, Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F12 medium (1:1 [v/ v]; #D8900; Sigma-Aldrich) containing 10% calf serum (#16170-078; Gibco BRL, Grand Island, NY) and 20 µg/ml gentamicin (#15750-060; Gibco BRL). The cells of each type were separately seeded at a density of 1 x 105 viable cells/ml in 48-well plates (Experiments 1 and 2; #150687; Costar, Cambridge, MA) or 30-ml bottles (Experiment 3; #83.1813.300; Sarstedt, Numbrecht, Germany) and cultured at 37.5°C in a humidified atmosphere of 5% CO2 and 95% air. To purify the stromal preparation, the medium was changed 6 hrs after plating, at which time selective attachment of stromal cells had occurred. Alternatively, because the epithelial cells attached 24 hrs to 48 hrs after plating, the medium in the epithelial cell culture was replaced 48 hrs after plating. The medium was changed every 2 days until confluency was reached. When the cells were confluent (6–7 days after the start of the culture), the medium was replaced with fresh DMEM and Ham’s F12 medium, supplemented with 0.1% bovine serum albumin (BSA; #A9056; Sigma-Aldrich), 5 ng/ml sodium selenite (#S1382; Sigma), 0.5 mM ascorbic acid (#A1417; Sigma-Aldrich), 5 µg/ml transferring, and 20 µg/ml gentamicin (#G-1397; Sigma-Aldrich). In Experiment 3, the cells were trypsinized from the bottles and suspended in calcium-free Hanks’ balanced salt solution (HBSS) supplemented with 0.1% BSA. The cells suspended in HBSS were then treated with phytoestrogens for intracellular Ca2+ mobilization measurement. The cells from 48-well plates were then exposed to various stimulators for the following experiments.

Experiment 1.
To determine the possible differential effect of phytoestrogen on epithelial and stromal cells, each type of cell was exposed to equol (10–8 M; #45405; Fluka Chemie GmbH, Buch, Switzerland), para-ethyl-phenol (10–8 M; #821290; Merck & Co., Inc., Gibbtown NJ), daidzein (10–8 M; #30405; Fluka Chemie GmbH), and genistein (10–8 M; #345834; Calbiochem-Novabiochem GmbH, Bad Soden, Germany) for 24 hrs. Estradiol 17ß (10–9 M; #75262; Fluka Chemie GmbH) was used as reference compound. Tumor necrosis factor-{alpha} (TNF-{alpha}; 6 x 10–11 M; Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) and oxytocin (10–6 M; #O4375; Sigma-Aldrich) were used as positive controls for stromal and epithelial cells, respectively (25, 31). An enzyme immunoassay measured PGF2{alpha} and PGE2 concentrations in culture medium.

Experiment 2.
To determine the intracellular mechanism of phytoestrogen action on the bovine endometrial epithelial and stromal cells, the cells were preincubated for 0.5 hrs with an ER antagonist (ICI-7{alpha},17ß-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol; 10–6 M; #129453-61-8; Tocris Cookson Inc, Ellisville, MO), a translation inhibitor (actinomycin D; 12.5 x 10–4 M; #114666; Calbiochem-Novabiochem GmbH), a protein kinase A (PKA) inhibitor (staurosporin; 10–7 M; #569398; Calbiochem, EMD Biosciences, San Diego, CA), and a phospholipase C (PLC) inhibitor (U73122; 10–6 M; #662035; Calbiochem, EMD Bioscience). After this pre-incubation, the cells were stimulated with equol (10–8 M), para-ethyl-phenol (10–8 M), and E2 as the reference compound (10–9 M). The two phytoestrogen metabolites are present in high concentrations, in conjugated form, in the blood plasma of cows that are fed diets rich in soy bean (18, 19). Unconjugated, active phytoestrogen metabolites are also present in the blood plasma of these cows in high concentrations. The same concentrations of phytoestrogens found in the plasma were used for treatment in the present in vitro study (18, 19). Concentrations of PGF2{alpha} in culture medium were measured by an enzyme immunoassay.

After the culture, the conditioned media were collected in tubes with 5 µl EDTA and 1% aspirin solution (pH 7.3; #A209; Sigma-Aldrich) and frozen until measurement of PGF2{alpha} and PGE2. Protein content was estimated by the method of Lowry et al. (32) and was used to standardize PGF2{alpha} and PGE2 concentrations. The results were expressed as ng/µg protein.

Experiment 3.
To determine the effect of phytoestrogens on intracellular Ca2+ mobilization, endometrial epithelial and stromal cells were exposed to equol (10–8 M, 10–7 M, and 10–6 M), para-ethyl-phenol (10–8 M, 10–7 M, 10–6 M), and E2 as the reference compound (10 –9 M, 10–8 M, and 10–7 M). The intracellular calcium mobilization was measured by the quantitative method described by Skarzynski and Okuda (33). In the method cell permeable form of the fluorescent Ca2+ indicator, Fura-2 (Fura-2 AM; #384-0583; Dojindo, Kumamoto, Japan) was used.

Intracellular Calcium Mobilization.
In the quantitative method, epithelial and stromal cells were trypsinized from the bottles and suspended in calcium-free HBSS supplemented with 0.1% BSA. Then, the cells were washed three times by centrifugation in calcium-free HBSS (5 mins; 100 g). Fura-2 AM, the lipophilic acetoxymethylester form of Fura-2, was dissolved in dimethyl sulfoxide to form a 1-mM stock solution, and 10 µl was added to 2-ml cell suspensions (5 µM final concentration) to preload the cells with dye. The cells were incubated for 30 mins at 37°C and then washed three times in calcium-free HBSS. After washing, the cells were postincubated for 30 mins in HBSS at room temperature to ensure full hydrolysis of the Fura-2 ester. Spectrofluorometric measurements were conducted in 1.5-ml samples continuously stirred in a quartz-glass cuvette and thermostatically maintained at 37°C. Fluorescence was monitored using a Shimadzu spectrofluorometer RF-5000 (Shimazu, Kyoto, Japan). In millisecond intervals, the intensity of fluorescence was measured in the cells treated with equol (10–8 M in the 60th second of the experiment, 10–7 M in the 120th second of the experiment, and 10–6 M in the 180th second of the experiment), para-ethyl-phenol (10–8 M in the 60th second of the experiment, 10–7 M in the 120th second of the experiment, and 10–6 M in the 180th second of the experiment), and E2 (10–9 M in the 60th second of the experiment, 10–8 M in the 120th second of the experiment, and 10–7 M in the 180th second of the experiment). Excitation and emission wavelengths were 340 nm and 490 nm, respectively, with slit widths of 5 nm for both wavelengths. Intracellular [Ca2+]i concentrations were calculated from the following equation:


where F is the fluorescence in the examined sample and KD is the dissociation constant for Fura-2–Ca2+ complex at 37.5°C is 2.24 x10–7 M. Maximum fluorescence (Fmax) was measured by maximum mobilization of the Fura-2–Ca2+ complex with phorbol 12-myristate 13-acetate (10–7 M; #P148; Sigma-Aldrich).

Hormone Determination.
Concentrations of PGE2 and PGF2{alpha} in the culture medium were determined with the enzyme immunoassays as previously described (31, 33). The PGE2 standard curve ranged from 0.39 ng/ml to 100 ng/ ml, and the ED50 of the assay was 6.25 ng/ml. The intra- and interassay coefficients of variation were 1.6% (n = 10) and 11.0% (n = 10), respectively. The PGF2{alpha} standard curve ranged from 0.016 ng/ml to 4 ng/ml, and the ED50 of the assay was 0.25 ng/ml. The intra- and interassay coefficients of variation were, on average, 11.3% (n = 10) and 7.1% (n = 10), respectively.

Statistical Analysis.
The data obtained from the Experiments 1 and 2 are shown as the mean ± SEM of values obtained in four separate experiments, each performed in triplicate. The statistical significance of differences between control and treated groups was assessed by one-way ANOVA followed by Bonferroni’s multiple comparison test. Patterns of Ca2+ mobilization in Experiment 3 were estimated by tests for repeated measures. All tests were performed by computer using Prism 4 software (GraphPad PRISM; GraphPad Software, Inc., San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effects of Oxytocin, Phytoestrogens, Their Metabolites, and E2 on PGF2{alpha} and PGE2 Production in Epithelial Cells.
Figure 1Go shows PGF2{alpha} (a) and PGE2 (b) production by epithelial cells in response to oxytocin, equol, para-ethyl-phenol, daidzein, genistein, and E2. Equol, para-ethyl-phenol, daidzein, and genistein (all 10–8 M) stimulated the secretion of PGF2{alpha} in epithelial cells (5.6-, 5.8-, 6.2-, and 6.0-fold, respectively; P < 0.001) compared with controls. Equol, para-ethyl-phenol, daidzein, and genistein (all 10–8 M) stimulated the secretion of PGE2 in epithelial cells (3.0-, 2.3-, 3.4-, and 3.7-fold, respectively; P < 0.001). At a concentration of 10 –9 M, E2 stimulated PGF2{alpha} and PGE2 secretion in epithelial cells (P < 0.05). Oxytocin, at a concentration of 10–7 M, stimulated the production of both PGs in epithelial cells (P < 0.001), which accounts for appropriate responsiveness of the cells.



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  		Figure 1. The effects of oxytocin (OT), equol, para-ethyl-phenol (P-e-phen), daidzein (Daidz), and genistein (Genist) on the production of prostaglandin (PG) PGF2{alpha} (a) and PGE2 (b) by bovine epithelial cells. Oxytocin (10–6 M), phytoestrogens, and their metabolites (all 10–8 M) were added 24 hrs before the end of culture. E2, estradiol 17ß. Asterisks indicate significant differences between control and treated groups (* P < 0.05, ***P < 0.001) as determined by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 4).

 
Effects of TNF-{alpha}, Phytoestrogens, Their Metabolites, and E2 on PGF2{alpha} and PGE2 Production in Stromal Cells.
Figure 2Go shows PGF2{alpha} (a) and PGE2 (b) production by stromal cells in response to TNF-{alpha}, equol, para-ethyl-phenol, daidzein, genistein, and E2. Equol, para-ethyl-phenol, daidzein, and genistein (all 10–8 M) stimulated thesecretion of PGF2{alpha} in stromal cells (2.3-, 2.4-, 2.4-, and 2.1- fold, respectively; P < 0.001). Equol, daidzein, and genistein (all 10–8 M) stimulated the secretion of PGE2 in stromal cells (1.8-, 1.5-, and 1.4-fold, respectively; P < 0.001). At a concentration of 6 x 10–11 M, TNF-{alpha} stimulated the production of both PGs in stromal cells (P < 0.001), which accounts for appropriate responsiveness of the cells.



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  		Figure 2. The effects of tumor necrosis factor-{alpha}, equol, para-ethyl-phenol, daidzein, and genistein on the production of prostaglandin (PG) PGF2{alpha} (a) and PGE2 (b) by bovine stromal cells. Tumor necrosis factor-{alpha} (TNF{alpha}; 6 x 10–11 M), phytoestrogens, and their metabolites (all 10–8 M) were added 24 hrs before the end of culture. Asterisks indicate significant differences between control and treated groups (*P < 0.05; **P < 0.01; ***P < 0.001) as determined by one-way ANOVA followed by Bonferroni’s multiple comparison test (n = 4).

 
Effects of Phytoestrogens on the PGF2{alpha} to PGE2 Ratio in Epithelial and Stromal Cells.
The basal PGF2{alpha} to PGE2 ratio in untreated cells (control) was 2.6 ± 0.2 and 0.2 ± 0.02 in epithelial and stromal cells, respectively. Treatment with phytoestrogens and their metabolites induced an increase in the PGF2{alpha} to PGE2 ratio in epithelial cells, but it did not affect the PGF2{alpha} to PGE2 ratio in stromal cells (Table 1Go). In epithelial cells, phytoestrogens and their metabolites significantly in-creased the PGF2{alpha} to PGE2 ratio from 2.6 ± 0.2 (control) to 5.7 ± 0.8 (daidzein), 5.0 ± 1.1 (genistein), 5.0 ± 0.4 (equol), and 7.4 ± 1.5 (para-ethyl-phenol), respectively (P < 0.05).


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  		Table 1. The Effects of Oxytocin, Tumor Necrosis Factor-{alpha}, Phytoestrogens, and Their Metabolites on the PGF2{alpha} to PGE2 Ratio in Epithelial and Stromal Cells
 
Effects of ICI, Actinomycin D, Staurosporin, and U73122 on Phytoestrogen and E2-Stimulated PGF2{alpha} Production in Epithelial and Stromal Cells.
The PKA inhibitor (staurosporin) and PLC inhibitor (U73122) did not affect the stimulative effect of active phytoestrogen metabolites equol (a) and para-ethyl-phenol (b) on PGF2{alpha} production by epithelial (Fig. 3Go) and stromal (Fig. 4Go) cells (P > 0.05). The stimulative effect of examined substances on PGF2{alpha} production by epithelial and stromal cells was reduced by the ER antagonist, ICI, and the translation inhibitor, actinomycin D (P < 0.001). The stimulative effect of E2 on PGF2{alpha} production by epithelial cells was reduced by all antagonists and inhibitors used (P < 0.05).



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  		Figure 3. The influence of an estrogen-receptor antagonist (i.e., ICI), a translation inhibitor (i.e., actinomycin D [Act-D]), a protein kinase A inhibitor (i.e., staurosporin), and a phospholipase C inhibitor (i.e., U73122) on equol-stimulated (a) and para-ethyl-phenol–stimulated (b) prostaglandin (PG) PGF2{alpha} production in bovine epithelial cells. The cells were preincubated for 0.5 hrs with ICI (10–6 M), actinomycin D (12.5 x 10–4 M), staurosporin (10–7 M), and U73122 (10–6 M) and then stimulated with equol (10–8 M) and para-ethyl-phenol (10–8 M). Asterisks indicate significant differences between control and treated groups (*P < 0.05, ***P < 0.001) as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test.

 


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  		Figure 4. The influence of an estrogen-receptor 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) on equol-mediated (a) and para-ethyl-phenol–mediated (b) prostaglandin (PG) PGF2{alpha} production in bovine stromal cells. The cells were preincubated for 0.5 hrs with ICI (10–6 M), actinomycin D (12.5 x10–4 M), staurosporin (10–7 M), and U73122 (10–6 M) and then stimulated with equol (10–8 M) and para-ethyl-phenol (10–8 M). Asterisks indicate significant differences between control and treated groups (***P < 0.001) as determined by two-way ANOVA followed by Bonferroni’s multiple comparison test.

 
Effects of Phorbol 12-Myristate 13-Acetate, Phytoestrogen Metabolites, and E2 on Intracellular Ca2+ Mobilization in Epithelial and Stromal Cells.
Neither equol nor para-ethyl-phenol (10–8 M, 10–7 M, and 10–6 M for both) caused the intracellular Ca2+ mobilization in epithelial and stromal cells (Fig. 5a and bGo; P > 0.05). Estradiol 17ß (10–9 M, 10–8 M, and 10–7 M) caused the intracellular Ca2+ mobilization in epithelial and stromal cells (Fig. 5cGo; P < 0.05). Phorbol 12-myristate 13-acetate (10–7 M) induced the intracellular Ca2+ mobilization in epithelial and stromal cells (Fig. 5a and bGo; P < 0.05), which accounts for appropriate responsiveness of the cells.



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  		Figure 5. The influence of equol (10–8 M, 10–7 M, and 10–6 M) (a) and para-ethyl-phenol (10–8 M, 10–7 M, and 10–6 M) (b) on the concentrations of intracellular calcium in epithelial cells (straight line) and stromal cells (dotted line) of bovine endometrium. Changes of calcium concentrations are shown as changes in the fluorescence of Ca2+ (Fura-2 AM) complex. Phorbol 12-myristate 13-acetate (10–7 M) was used as a positive control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The present study demonstrated that, in bovine endometrium, phytoestrogens and their metabolites mainly stimulated PGF2{alpha} production in epithelial cells. Moreover, our results indicated that phytoestrogens and their active metabolites act mainly through a genomic, ER-dependent mechanism without intracellular calcium mobilization.

The main source of PGF2{alpha} in bovine endometrium is known to be epithelial cells, whereas stromal cells produce approximately 10 times more of luteotropic PGE2 in comparison to epithelial cells (22, 24, 25). In the present study, phytoestrogens and their metabolites preferentially stimulated PGF2{alpha} synthesis in epithelial cells (560%–620% of control) compared with stromal cells (210%–240% of control) and moderately, but significantly, increased PGE2 production in both epithelial cells (230%–370%) and stromal cells (140%–180%). In comparison, E2 stimulated PGF2{alpha} and PGE2 production (140% for both PGs) only in epithelial cells (P < 0.05). Our data showed that the basal PGF2{alpha} to PGE2 ratio in epithelial cells was 10 times higher than the PGF2{alpha} to PGE2 ratio in stromal cells. Moreover, treatment with phytoestrogens and their metabolites resulted in a 2.5 to 3.5 time increase in the PGF2{alpha} to PGE2 ratio in epithelial cells. In contrast, the same treatment with phytoestrogens did not modify the PGF2{alpha} to PGE2 ratio in stromal cells. Moreover, phytoestrogen metabolites have an enhancing effect on PGF2{alpha} production in oxytocin-treated epithelial cells. This synergic effect of phytoestrogens and oxytocin may additionally increase the ratio of luteolytic PGF2{alpha} to luteotropic PGE2 production in bovine endometrium. The results indicate that phytoestrogens and their metabolites differentially modulate PG synthesis in a cell-specific manner, increasing both PGs without altering the PGF2{alpha} to PGE2 ratio in stromal cells and directing the biosynthetic pathway toward PGF2{alpha} in epithelial cells. Thus, it appears that phytoestrogens and their metabolites mainly modulate the PGF2{alpha} to PGE2 ratio in epithelial cells. In the endometrium, a proper PGF2{alpha} to PGE2 ratio is essential for maintaining an optimum uterine environment for embryo implantation and development (21, 34). During embryo development and implantation, the PGF2{alpha} to PGE2 ratio decreases due to an increase of PGE2 production (34). The increased PGE2 stimulates P4 synthesis in the confidence limits (35). Based on the stronger phytoestrogen-dependent stimulation of PGF2{alpha} compared with PGE2 production in epithelial cells observed in the present study, we assume that phytoestrogens cause premature luteolysis, leading to embryonic loss during early pregnancy in cattle. This supposition has been supported by our recent in vivo and in vitro findings (19). We have shown that soy bean–derived phytoestrogens and their metabolites act as endocrine disruptors, leading to disruption of the reproductive processes and temporal infertility of cows. Phytoestrogens and their active metabolites disrupt the ratio of PGE2 to PGF2{alpha}, which leads to the nonphysiologic production of luteolytic agent in cattle during the estrous cycle and pregnancy (19).

Estrogens exert their physiologic effects in target cells by genomic (36) and nongenomic pathways (37), as shown by the results obtained in Experiment 2. The genomic pathway involves activation of ERs and modification of gene expression (38–41). The genomic mechanism of estrogen action depends on the presence of {alpha} and ß, two types of ER (42). It has been proven that some phytoestrogens have higher affinity to ERß than to ER{alpha} (8, 43). Phytoestrogens and their active metabolites may compete with endogenous E2, thus disturbing the processes influenced by E2. The stimulatory effect of E2 on the synthesis of both PGs in epithelial cells was reduced by an ER antagonist (ICI) and a translation inhibitor (actinomycin D) supporting its genomic action. In the present study, ICI and actinomycin D also blocked the stimulatory effect of equol and para-ethyl-phenol on PGF2{alpha} synthesis in endometrial epithelial and stromal cells. The study of Dubey et al. (14) revealed that a selective ER antagonist, ICI, blocked the E2 influence on endothelial cells of the blood vessels, suggesting genomic action mediated by ER{alpha}. The results of Wang et al. (26), and Wang and Kurzer (27) proved that phytoestrogens act via binding to both ER{alpha} and ß. Therefore, we assume that, owing to phytoestrogen structural similarity to E2, they may elicit or selectively modulate genomic estrogenic responses by binding to both ERs, like endogenous E2.

In the nongenomic pathway of estrogen action, PLC and PKA are the most important compounds of the intracellular second messenger system. The results of the present study contrasted with findings of some research groups and agreed with others. Morley et al. (28), Katzenellenbogen (29), and Smith (30) found that endogenous E2 can act via a nongenomic way, especially via PKA action and intracellular calcium (Ca2+) mobilization. Szego (44) found that endogenous steroids induced signaling pathways connected with membrane-bound enzymes such as PLC and PKA, which lead to the intracellular increase of cAMP and calcium mobilization (45, 46). In our study, the stimulatory effect of E2 on the synthesis of both PGs in epithelial cells was reduced by a PLC inhibitor, U73122, and a PKA inhibitor, staurosporin, supporting its non-genomic action. On the other hand, Dubey et al. (14) found that genistein inhibited mitogen-activated protein kinase activity and mRNA for transforming growth factor–ß and integrin {alpha}vß3 expression (47), tyrosine kinase activity (48), phospholipase D activity (49), and PLC-dependent intra-cellular calcium release (50). However, in the present study, neither the PLC inhibitor nor the PKA inhibitor (inhibitors of nongenomic pathways and second messengers) inhibited equol-mediated and para-ethyl-phenol–mediated stimulation of PGF2{alpha} synthesis in epithelial and stromal cells. These results suggest the lack of a nongenomic mechanism of phytoestrogen metabolites action on the PG synthesis in bovine endometrium, in contrast to endogenous E2.

In conclusion, the present study demonstrated that phytoestrogens stimulate both PGF2{alpha} and PGE2 in both cell types of bovine endometrium via an ER-dependent genomic pathway. However, because phytoestrogens preferentially stimulated PGF2{alpha} synthesis in epithelial cells of bovine endometrium, they may disrupt uterus function by altering the PGF2{alpha} to PGE2 ratio. This action of phytoestrogens on PGF2{alpha} may account, at least in part, for the reproductive disorders observed in ruminants fed diets that are rich in soy.


   Acknowledgments
 
We thank Dr. Seiji Ito of Kansai Medical University, Osaka, Japan, for PGF2{alpha} and PGE2 antiserum and Dainippon Pharmaceutical Co., Ltd., Osaka, Japan, for recombinant human TNF-{alpha} (HF-13).


   Footnotes
 
This work was supported by Grants-in-Aid for Scientific Research from the Polish Ministry of Scientific Research and Information Technology (KBN 5P06K 003 21) and the Japan Society for the Promotion of Science (B14360168). I.W.-P. was supported by the Japanese-Polish Joint Research Project under the agreement between the Japan Society for the Promotion of Science and the Polish Academy of Sciences.

Received for publication December 8, 2004. Accepted for publication February 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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