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Experimental Biology and Medicine 230:189-199 (2005)
© 2005 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Soybean-Derived Phytoestrogens Regulate Prostaglandin Secretion in Endometrium During Cattle Estrous Cycle and Early Pregnancy

Izabela Woclawek-Potocka*, Mamadou M. Bah{dagger}, Anna Korzekwa*, Mariusz K. Piskula{dagger}, Wieslaw Wiczkowski{dagger}, Andrzej Depta{ddagger} and Dariusz J. Skarzynski*,1

* Department of Reproductive Immunology, {dagger} Department of Food Technology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn, Poland; and {ddagger} Faculty of Veterinary Medicine, Warmia and Mazury University, Olsztyn, Poland

1To whom requests for reprints should be addressed at Department of Reproductive Immunology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyne, Poland. E-mail: skadar{at}pan.olsztyn.pl


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 PGF2{alpha} (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 PGF2{alpha}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 PGF2{alpha} to PGE2, which leads to high, nonphysiological production of luteolytic PGF2{alpha} in cattle during the estrous cycle and early pregnancy.

Key Words: phytoestrogens • estrous cycle • cow • prostaglandins


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The role of estrogenic substances derived from plants has been studied for almost half a century (reviewed in 1–3). Environmental estrogens are divided into two main groups: phytoestrogens and xenoestrogens (4). Xenoestrogens are man-made synthetic products, whereas phytoestrogens are derived from plants. There is some evidence that consumption of soy diets containing phytoestrogens has some positive effects on human and animal health. Phytoestrogens are thought to reduce the risk of mammary cancer (5, 6), act as antioxidants (7), and modify certain enzyme activities (8). Moreover, the consumption of soybeans by postmenopausal women has been associated with other health benefits, such as reducing the risk of cardiovascular disease (9, 10) and cancer (11), stopping the progression of atherosclerosis (12, 13), and having positive effects on hot flashes, vaginal symptoms, cognitive function or dementia (14), and bone preservation (15). On the other hand, these substances also have some hazardous effects, especially in animals fed with pasture rich in phytoestrogens (16, 17). Ingestion of clover pasture rich in plant estrogens was shown to cause infertility in cattle (18). There is increasing evidence that phytoestrogens can disrupt reproductive efficiency in various species, including humans (19, 20), rats (21), and cows (22).

Phytoestrogens may act like antagonists or agonists of endogenous estrogens (20, 23–25). Endogenous estrogens control the estrous cycle in ruminants influencing prostaglandin (PG) synthesis (reviewed in 26). For example, the removal of 17-ß-estradiol (E2) 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 (27). On the other hand, administration of E2 to heifers on Day 13 of the cycle initiated luteolysis by increasing the PGF2{alpha} concentration (28). The role of PGs in the reproductive processes of many species is well established (29). In ruminants, PGF2{alpha} is the major luteolytic agent (30), whereas PGE2 has luteoprotective and anti-luteolytic properties (31, 32). Therefore, the development and maintenance of the corpus luteum (CL) as well as establishment of pregnancy may depend on the ratio of luteolytic PGF2{alpha} to luteotropic PGE2 (33). In view of the structural and functional similarities of phytoestrogens and endogenous estrogens, we suspect that these plant-derived substances modulate prostaglandin synthesis in the bovine endometrium.

The fodder commonly used for feeding dairy cattle contains phytoestrogens, such as genistein, daidzein, formonentin, and biochanin A (34). Lundh et al. (35) showed that, in cows and ewes, daidzein and genistein present in the fodder are immediately converted in the rumen to equol and p-ethyl-phenol, respectively. The concentration of daidzein and genistein decreases within 1 hr after feeding, whereas equol and p-ethyl-phenol are present in the blood of cows for many hours after feeding (35). Although metabolism of phytoestrogens from synthetically prepared fodder that is rich in phytoestrogens has been thoroughly investigated by Lundh et al. (34, 35), little is known about the effects of feeding cattle with fodder rich in phytoestrogens derived from natural soybean. Therefore, the present study was undertaken to identify which metabolites of phytoestrogens are present in the blood of cows fed a diet rich in soybean, and what phytoestrogen concentrations are needed to have an effect on the bovine reproductive tract. Furthermore, we examined whether phytoestrogens identified in soybean and their metabolites regulate PG output from the bovine endometrium in vitro and if they influence the PGF2{alpha} to PGE2 ratio during the estrous cycle in cattle.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Collection of Endometrial Tissues.
The study consisted of in vivo experiments and an in vitro experiment. The in vivo experiment was performed on mature, normally cycling Holstein/Polish black and white (75% and 25%, respectively) cows (n = 22) and heifers (n = 10). All experimental procedures concerning live animals were approved by the Local Animal Care and Use Committee (agreement 4/2001/N).

For the in vitro experiment, bovine uteri were obtained at a local abattoir within 30 min after exsanguination and were transported, on ice, to the laboratory within 1 hr. Bovine uteri were classified into five stages of the estrous cycle (early luteal I, Days 1–4; early luteal II, Days 5–8; mid luteal, Days 8–12; late luteal, Days 12–15; and follicular, Days 18–21). The stages of the estrous cycle were estimated by macroscopic observation of the ovaries and uterus (36). The uterine horns were separated from each other and from the remaining tissue.

Endometrial Tissue Culture.
Endometrial strips were washed three times in sterile saline containing 100 IU/ml penicillin and 100 µg/ml streptomycin. The tissue was then cut into small pieces (approximately 20–30 mg) with a scalpel and subsequently washed in sterile saline. The individual endometrial tissues were placed in culture glass tubes (12 x 75 mm) containing 3 ml of culture medium (Dulbecco’s Modified Eagle’s Medium and Ham’s F-12 medium 1:1 [volume/volume {v/v}]; Sigma Chemical Company, St. Louis, MO; #D8900) supplemented with 0.1% BSA (Boehringer Mannheim GmbH, Mannheim, Germany; #735078), 100 IU/ml penicillin, and 100 µg/ml streptomycin. The tissues were incubated in a shaking water bath at 37°C as described previously (36). The media were continuously gassed with 5% CO2 in air during incubation.

Experiments.
Experiment 1. Effects of soy-derived phytoestrogens on reproductive efficiency.
The experiment was carried out on two animal farms, with different feeding systems. On one farm in Zalesie, 12 cows were fed a standard diet (Herd Z), and on the other farm in Watkowice, 12 animals were fed a diet containing soybean (Herd W). All experimental cows had given birth to healthy offspring in September and October of the previous year (2002). After calving, the cows were fed during lactation with the scheme shown in Table 1Go.


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  		Table 1. Comparison of Feeding Systems in the Z and W Herdsa
 
In the next year, all cows of both herds were impregnated by artificial insemination (AI) with the semen of the same bull, called Macassar (no. 95113-4-5). The semen was purchased from the Station of Animal Insemination and Breeding in Bydgoszcz, Poland. The onset of the estrus was confirmed by standing behavior and determined by farm workers as well as by the inseminating veterinarian via rectal examination. The estrus was taken as Day 0 of the estrous cycle. Only the cows with behavioral signs of estrus underwent artificial insemination after positive rectal examination. After inseminations, the cows were observed until early October 2003. During the observation, blood samples were taken from the jugular vein every 3 months and the condition of reproductive organs was examined rectally. At the end of the experiment (early October 2003), urine samples were also taken from the observed cows. The blood plasma was separated by centrifugation (2000 g, 10 min, 4°C) and stored at –20°C until determination of hormones was made. Concentrations of free and conjugated phytoestrogens in plasma and urine of cows of the two examined herds were determined by high-performance liquid chromatography (HPLC).

Experiment 2. Effect of soy-derived phytoestrogens on prostaglandin secretion during early pregnancy.
The objectives of this experiment were to examine (i) the influence of a high soybean diet on the concentrations of P4, PGFM (a metabolite of PGF2{alpha} —13,14, keto PGF2{alpha}), and PGE2 in the blood plasma of experimental heifers after artificial insemination and (ii) the possible influence of phytoestrogens present in the soybean on the reproductive efficiency of heifers.

The experiment was carried out on Herd Z from July to September 2004. We chose 10 normally cycling Holstein/ Polish black and white (75% and 25%, respectively) heifers (18–20 months of age and 400–450 kg body weight). The animals in Herd Z were fed a standard diet (Table 1Go). Two weeks after weighing and choosing the animals for the experiment, the estrus was synchronized using implants of a progesterone analog (Crestar; Intervet, Boxmeer, Holland) as described previously (37). When Crestar was removed the animals (n = 10) were divided into two groups: control (n = 5) and soybean diet (n = 5). Control animals were continuously fed a standard diet (Table 1Go, Herd Z). Five other heifers were fed 2.5 kg/day of soybean instead of bruised sunflower grain and wheat bran until Day 21 of either the estrous cycle or pregnancy. The onset of estrus was confirmed by standing behavior as determined by workers on the farm as well as by the veterinarian via rectal examination. The estrus was taken as Day 0 of the estrous cycle. Seventy–two and 84 hr after Crestar removal, each heifer was inseminated twice with the semen of a bull called Math TV (no. 29960031953). The semen was a gift from Union Nord-Ouest Genetique (UNOG, Lisiex, Bosc-Berenger; St. Saens, France). Only the heifers with behavioral signs of estrus underwent AI after positive rectal examination. The blood samples were collected via puncture of the jugular vein on Days 0, 2, 5, 8, 12–18, and 21 of either pregnancy or estrous cycle. The blood plasma was separated by centrifugation (2000 g, 10 min, 4°C) and stored at –20°C until determination of hormones was made. Concentrations of free and conjugated phytoestrogens in plasma were determined by HPLC.

Experiment 3. Determination of effective dose of soy-derived phytoestrogen in vitro.
Endometrial slices from early luteal I stage (Day 1–4 of the estrous cycle) were treated for 24 hr with various concentrations (0.1 nM to 1 µM) of equol (#45405, Fluka Chemie GmbH, Seelze, Germany), p-ethyl-phenol (#821290, Merck & Co., Inc., Whitehouse Station, NJ), daidzein (#30405 Fluka Chemie), genistein (#345834, Calbiochem-Novabiochem GmbH, Darmstadt, Germany) or E2 (#75262, 1 nM; Fluka Chemie), and tumor necrosis factor-{alpha} (TNF{alpha}, 0.6 nM; Dainippon Pharmaceutical Co. Ltd., Osaka, Japan) for a positive control. After 24 hr of incubation, the conditioned media were collected in tubes with 10 µl EDTA, 1% aspirin (#A2093; Sigma), solution (pH 7.3), and frozen at –20°C until measurement of PGF2{alpha} The conditioned media were examined for the concentrations of PGF2{alpha} by enzyme immunoassay (EIA).

Experiment 4. Effect of soy-derived phytoestrogens on prostaglandin output at different stages of the estrous cycle in vitro.
Endometria were taken from cows at five stages of the estrous cycle: early-I luteal (n = 4), early-II luteal (n = 4), midluteal (n = 4), late luteal (n = 4), and follicular (n = 4). Endometrial slices were exposed to daidzein and genistein and their metabolites, equol and p-ethyl-phenol (10 nM each) or E2 (1 nM), and TNF{alpha} (0.6 nM) for a positive control (38). The phytoestrogen concentration of 10 nM was chosen because a preliminary experiment showed that it was the most effective dose. After 24 hr of incubation, the conditioned media were collected in tubes with 10 µl EDTA, 1% aspirin (#A1093; Sigma), solution (pH 7.3), and frozen at –20°C until measurement of PGF2{alpha} and PGE2. The tissues were blotted with filter paper and weighed to obtain the concentration per gram tissue.

Analytical Methods.
Determination of phytoestrogens and their conjugates in soybean.
Phytoestrogens were extracted from soybean and identified by HPLC-UV-mass spectroscopy (MS). Pulverized soybean (ca. 0.15 g) was defatted with n-hexane by subsequent sonication and centrifugation (5 x 1 ml). Defatted soy was extracted with 1 ml of 80% methanol containing 0.3 M hydrochloric acid by a 30-sec sonication. The mixture was vortexed for 30 secs, again sonicated and vortexed, and centrifuged for 5 min (5000 g at 4°C). The supernatant was collected in a 5-ml volumetric flask. That step was repeated five times. The obtained extract was directly submitted to HPLC analysis. Standard compounds (daidzein, daidzin, genistein, genistin, glycitein) were dissolved in 80% methanol containing 0.3 M hydrochloric acid, and their concentration was confirmed by UV measurement. Concentration of the daidzin 6-OMalGlc, 6-OAcGlc conjugates, genistin 6-OMalGlc, 6-OAcGlc conjugates, and glycitin were calculated from the daidzin, genistin, and glycitein standard curves, respectively.

Chromatographic determinations were done on a Shimadzu HPLC LC-10 gradient system (Shimadzu, Kyoto, Japan) consisting of a system controller, two pumps, UV detector set at 254 nm, MS detector (QP8000{alpha}), autosampler with 5-µl injection loop, and column oven. All chromatographic determinations were performed at 35°C, at a flow rate of 0.2 ml/min on a C18(2) Luna 3µ column, 150 x 2 mm (Phenomenex, Torrance, CA). The mobile phase was composed of a mixture of solvent A (water, with 0.05% formic acid [v/v]) and solvent B (acetonitrile). Gradients were as follows: 10%B, 28%B, 37%B, 60%B, 60%B, 10%B, 10%B at gradient times tG = 0, 14, 44, 49, 54, 55, and 75 mins, respectively. Identities were confirmed by mass spectrometry. Mass spectrometer settings were curve desolvation line (CDL) temperature, 160°C; CDL voltage, –70 V; probe voltage, –2,5 kV; defragmentation voltage, –50 V; nitrogen as nebulizer gas flow at 2.8 ml/ min. Three replications were done for each analysis.

Determination of phytoestrogens and their conjugates in bovine plasma and urine.
Plasma and urine concentration of phytoestrogens and their metabolites were measured as described previously (39). Nonconjugated daidzein, genistein, or their metabolites were determined on HPLC after extraction from blood plasma. To 50 µl of plasma, 50 µl of 0.2 M sodium acetate buffer, pH 5, and 900 µl of methanol/acetic acid (100:5, v/v) were added. The mixture was vortexed for 30 secs, sonicated for 30 secs, again vortexed for 30 secs, and centrifuged for 5 mins at 4°C and 5000 g. The supernatant was diluted with 100 mM of lithium acetate (1:1, v/v), centrifuged for 2 mins at 4°C and 5000 g, and 20 µl was injected onto an HPLC column (TSKgel ODS-80TS, 5 µm, 150 x4.6 mm; TOSOH, Tokyo, Japan). The flow of the mobile phase, composed of water/ methanol/acetic acid (58:40:2, v/v/v) containing 50 mM of lithium acetate, was 0.9 ml/min. The eluate was monitored with an amperometric detector (ICA-3062; TOA, Tokyo, Japan) with the working potential set at +950 mV. When necessary, samples were diluted with the mobile phase before HPLC analysis.

Enzymatic hydrolysis of the conjugates of daidzein, genistein, and their metabolites and determination of concentrations of released free forms in plasma and urine.
Cow plasma and urine samples (50 µl) were mixed with 50 µl of sulfatase type H-5 solution in 0.2 M acetate buffer, pH 5 (the preparation contained 500 U of ß-glucuronidase per 25 U of sulfatase), and the mixture was incubated at 37°C in a shaking water bath for 1 hr. Daidzein, genistein, and their metabolites released during the incubation and their nonconjugated forms present in plasma before the hydrolysis were extracted with 900 µl of methanol/acetic acid (100:5, v/v) and determined as described above. The result was the total plasma concentration of daidzein, genistein, and their respective metabolites.

Blood biochemical analyses.
Blood was collected from the jugular vein. Serum activities of alkaline phosphatase (ALP), alanine aminotransferase (ALT), and asparagine aminotransferase (AST) were determined using kits (Pointe Scientific, Lincoln Park, MI). The levels of electrolytes (phosphorus [P+], magnesium [Mg2+], calcium [Ca2+]) were determined with an electrolyte analyzer (EasyLyte; Medica, Bedford, MA) and ion-selective electrodes. Glucose content was measured by the oxidase method. Total protein content was measured with a kit (Alpha Diagnostics, San Antonio, TX). Cholesterol content was measured enzymatically with a kit (Pointe Scientific). Colorimetric and kinetic assays were done with a spectrophotometer (UV/VIS s 330; Marcel Euro, Marcel Sp. z.o.o, Warszawa, Poland).

Determination of Hormones.
Progesterone concentrations in plasma samples were assayed using a direct EIA as described previously (37). The P4 standard curve was produced for P4 concentrations ranging from 0.39 pg/ml to 25 ng/ml. The intra- and interassay coefficients of variation averaged 6.6% and 8.4%, respectively.

The concentrations of PGFM in the plasma samples were determined with a direct EIA, as described previously (37). The anti-PGFM serum (WS4468-5) was donated by Dr. W.J. Silvia, University of Kentucky, Lexington, KY. The PGFM standard curve was produced for PGFM concentrations ranging from 32.5 pg/ml to 8000 pg/ml. The intra- and inter-assay coefficients of variation were, on average, 7.6% and 10.4%, respectively.

The concentrations of PGE2 in the blood samples and in the culture medium were determined by a direct EIA test as described previously (37). The anti-PGE2 serum was donated by Dr. S. Ito, Kansai Medical University in Osaka, Japan. Cross-reactivities of the anti-PGE2 serum, determined by measuring the inhibition of binding of peroxidase-labeled PGE2 to this antiserum, were as follows: PGE2, 100%; PGE1, 18%; PGJ2, 14%; PGA1, 10%; 15-keto PGE2, 8.8%; PGB2, 6.7%; PGA2, 4.6%; PGD2, 0.13%, and PGF2{alpha}, 2.8%. For blood plasma samples, the PGE2 standard curve was produced for PGE2 concentrations ranging from 0.07 ng/ml to 20 ng/ml. For medium samples, the standard curve was produced for PGE2 concentrations ranging from 0.39 ng/ml to 100 ng/m. The intra- and interassay coefficients of variation were, on average, 6.9% and 9.7%, respectively.

The concentration of PGF2{alpha} in the culture medium was determined with the direct EIA test as described previously (38). The PGF2{alpha} standard curve ranged from 0.016 ng/ml to 4 ng/ml. The intra- and interassay coefficients of variation were, on average, 7.1% and 11.3%, respectively.

Statistical Analysis.
Least squares means and SEM were determined in the preliminary in vivo observations. Differences in biochemical indexes in the blood were analyzed using Student’s t test (GraphPad PRISM Version 4.00; GraphPad Software, San Diego, CA). The differences of mean number of cover and the percentage of the pregnancy were analyzed using an X2 test (GraphPad PRISM). Plasma concentrations of hormones in Experiment 2 were analyzed using ANOVA with repeated measures (GraphPad PRISM). Prostaglandins (PGFM, PGE2) and P4 in the jugular plasma samples, collected during the estrous cycle, were analyzed with a repeated measures design approach with treatments (soy-been fed vs. control heifers) and time of sample collection (days of the cycle) being fixed effects, with all interactions included. All analyses were done using repeated measures ANOVA tests followed by Bonferroni’s multiple comparison test (GraphPad PRISM; P < 0.05 was considered significant). The obtained data from the in vitro experiments (the concentrations of PGE2 and PGF2{alpha} in the cultured media) were exposed as the mean ± SEM of values obtained in 3–4 separate experiments, each performed in triplicate. The statistical significance of differences between controls and treated groups was assessed by one-way ANOVA, followed by the Dunnett comparison test (GraphPad PRISM).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Experiment 1.
Effects of soy-derived phytoestrogens on reproductive efficiency.
The total phytoestrogen content of fodder used to feed cows from Herd W was approximately 1900 µg/g (Fig. 1bGo). The phytoestrogens consisted mainly of daidzein and genistein glucosides. The same phytoestrogens were found in the fodder used to feed cows from Herd Z, but the total phytoestrogen content was smaller than 300 µg/g. The cows fed soybeans (Herd W) had significant levels of p-ethyl-phenol and equol in urine (365 ± 67 µM and 160 ± 24 µM, respectively) and in the plasma (1.6 ± 0.31 µM and 1.2 ± 0.28 µM, respectively; Fig. 1cGo). In contrast, no phytoestrogens were detected in urine or plasma of cows fed the standard diet (Herd Z). The phytoestrogen metabolites present in plasma and urine formed glucuronide and/or sulfate conjugates.



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  		Figure 1. ECD-high–performance liquid chromatography (HPLC) chromatograms: phytoestrogens in the soybean–containing fodder (a), phytoestrogens standards used in HPLC analysis (b), phytoestrogens in the plasma of cows fed with silage with soybean (c).

 
Blood analyses and other characteristics of Herds Z and W are compared in Table 2Go. One year after the beginning of observations, the pregnancy rate of cows fed the standard diet (100%) was significantly greater (P < 0.01) than that of cows fed the diet based on soybeans (60%). The mean number of cover for experimental cows (Herd W) differed significantly from the mean number of cover for control cows (4.0 ± 0.78 vs. 2.2 ± 0.33; P < 0.01). Moreover, plasma concentrations of total cholesterol, protein, and ALT activity were higher in the cows fed with soybeans (Herd W) compared with cows fed with standard diet (P < 0.05).


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  		Table 2. Effect of Different Feeding Schemes, Standard Diet (Herd Z) and Diet with Soybean (Herd W), on the Mean Number of Cover, Percent of Pregnancy, Glucose, Cholesterol, Phosphorus (P–), Magnesium (Mg2+), Calcium (Ca2+), Total Protein Concentrations, and Activity of Alanine Aminotransferase (ALT), Asparagine Aminotransferase (AST), Alkaline Phosphatase (ALP) in Seruma
 
Experiment 2.
Effect of soy-derived phytoestrogens on prostaglandin secretion during early pregnancy.
In the serum of soybean-fed heifers, only the conjugated forms of p-ethyl phenol and equol were found in high concentration (Fig. 2Go). Phytoestrogens and their metabolites in the blood of control animals were almost undetectable. Only p-ethyl-phenol was found in the blood of the control animals, and its concentration was low (0.45 ± 0.078 µM; data not shown). The pregnancy rates of the heifers fed the high-soy diet (3/5) and the heifers fed the standard diet (4/5) were not significantly different as determined by the presence of CL in the ovary and a P4 concentration higher that 2 ng/ml at Day 21 after insemination (Fig. 3Go). The concentrations of PGFM in the blood of heifers fed soybeans were significantly higher throughout the experimental period than those in the control heifers (P < 0.01; Fig. 4Go). The higher levels of PGFM were positively correlated with equol and p-ethyl phenol concentrations in the blood (P < 0.001). The differences of PGE2 concentrations between the two groups of heifers were not significantly different (Fig. 5Go; P > 0.05). PGE2 concentrations in both examined groups of heifers increased from Day 8 through Day 12 of the experiment from 0.49 ± 0.085 ng/ml to 1.634 ± 0.35 ng/ml, remaining at this high level in pregnant animals and decreasing to 0.43 ± 0.04 ng/ml in nonpregnant heifers (data not shown).



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  		Figure 2. Concentration of equol (a) and para-ethyl-phenol (b) in the peripheral blood plasma of heifers fed with soybean (2.5 kg soybean/ animal/day; n = 5) at 21 days after artificial insemination.

 


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  		Figure 3. Concentration of progesterone in the peripheral blood plasma of heifers fed with standard diet (a) and 2.5 kg/day/animal of soybean (b).

 


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  		Figure 4. Concentration of a metabolite of prostaglandin F2{alpha}-13,14, keto PGF2{alpha} (PGFM) in the blood plasma of heifers fed standard diet (a) and 2.5 kg/day/animal of soybean (b).

 


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  		Figure 5. Effects of various concentrations (0.1–1000 nM) of phytoestrogens and their metabolites on PGF2{alpha} production by bovine endometrial slices. Daidzein (a), genistein (b), equol (c), and p-ethyl-phenol (d), estradiol-17ß (E2; 1 nM) or tumor necrosis factor-{alpha} (TNF{alpha}; 0.06 nM) were added to the medium for 24 hr after 1 hr of preincubation. 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 the Dunnett comparison test.

 
Experiment 3.
Determination of the effective dose of the soy-derived phytoestrogens in vitro.
Both daidzein and genistein and their metabolites, equol and p-ethyl-phenol, stimulated PGF2{alpha} secretion by bovine endometrium during the early Stage I (P < 0.01; Fig. 5Go). The concentration of four compounds that gave the most significant increase (P < 0.001) was 10 nM. Therefore, a concentration of 10 nM was chosen for further studies.

Experiment 4.
Effect of soy-derived phytoestrogens on prostaglandin output at different stages of the estrous cycle in vitro.
Phytoestrogens and their metabolites stimulated the secretion of PGF2{alpha} (Fig. 6Go) and PGE2 (Fig. 7Go) in different stages of the estrous cycle in the bovine endometrium. PGF2{alpha} output was strongly stimulated (approximately 6-fold), whereas PGE2 output was stimulated by only about one third this amount (Table 3Go).



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  		Figure 6. Effects of equol (eq), p-ethyl-phenol (p-e-p), daidzein (daidz), and genistein (genist) on PGF2{alpha} output from the cultured bovine endometrium at (a) early luteal phase I (Days 1–4 of the estrous cycle), (b) early luteal phase II (Days 5–8 of the estrous cycle), (c) midluteal phase (Days 8–12 of the estrous cycle), (d) late luteal phase (Days 15–18 of the estrous cycle), and (e) follicular phase (Days 18–21 of the estrous cycle). Phytoestrogens and their metabolites (10 nM), E2 (1 nM) and TNF{alpha} (0.06 nM) were added 24 hr 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 the Dunnett comparison test.

 


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  		Figure 7. Effects of equol (eq), p-ethyl-phenol (p-e-p), daidzein (daidz), and genistein (genist) on PGE2 output from the cultured bovine endometrium at (a) early luteal phase I (Days 1–4 of the estrous cycle), (b) early luteal phase II (Days 5–8 of the estrous cycle), (c) midluteal phase (Days 8–12 of the estrous cycle), (d) late luteal phase (Days 15–18 of the estrous cycle), and (e) follicular phase (Days 18–21 of the estrous cycle). Phytoestrogens and their metabolites (10 nM), E2 (1 nM), and TNF{alpha} (0.06 nM) were added 24 hr 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 the Dunnett comparison test.

 

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  		Table 3. Effects of Equol and p-Ethyl-Phenol on the PGF2{alpha} and PGE2 Output in Endometrial Slices in Various Phases of the Estrous Cycle (Fold of Stimulation); Fold of Stimulation Was Defined on the Basis of Data from the Saline-Treated Slices (Control) in Each Phase of the Estrous Cycle; the Data are Shown as the Mean ± SEM from Four Experiments
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We found large amounts of daidzein and genistein in the soybean commonly used for feeding dairy cattle. These phytoestrogens occur in plants as glycosides and are hydrolyzed in the rumen by microorganisms (40). Daidzein is metabolized in the rumen to equol, whereas genistein is metabolized to p-ethyl-phenol (35). High concentrations of both of these metabolites were found in plasma and urine. Cattle fed the high-soy diet (Herd W) had higher serum concentrations of cholesterol, protein, and ALT than cattle fed the diet without soybean (Herd Z). These high concentrations may be due to the high total fat and protein content in the soy diet. However, all the serum constituents that were examined were in their normal physiological ranges, showing that the examined animals were in proper condition without any metabolic disorders.

The high-soy diet decreased the pregnancy rate. This confirms the findings of many other studies that phytoestrogens can disturb reproductive processes on many different regulatory levels (reviewed in 41). Phytoestrogens can inhibit hypophyseal luteinizing hormone secretion (42). This causes a decrease of progesterone production, which in turn leads to high abortion rate (43). The decrease of pregnancy rate can also be attributed to phytoestrogen-dependent inhibition of endogenous estrogens production leading to disturbances in follicle development and lack of estrus (4, 20). Therefore, phytoestrogens may disturb estrus and ovulation through their effects on the central nervous system. Moreover, high concentrations of estrogenic substances on the day of insemination can be associated with early embryonic loss (44). High productivity of dairy cows may lead to a higher insemination rate (45). A high protein diet often reduces reproductive efficiency in cows (46). We hypothesize that phytoestrogens decrease fertility in cows by modulating the production of prostaglandins. As shown in Experiments 1 and 2, the high-soy diet significantly decreased the rate of successful pregnancies as well as increased the mean insemination rate and PGFM concentration in the serum of soy-fed animals.

Phytoestrogens inhibit the binding of (H3)-E2 or (H3)-organon to their respective receptors, but the relative affinities of (H3)-E2 and (H3)-organon are lower than those of E2 (20, 47, 48). The affinities of phytoestrogens for estrogen receptors are only 0.1% to 1% of those of circulating estrogens (E2 or estrone) (1, 23, 49). Thus, the many biological effects attributed to phytoestrogens may be due to their relatively high concentrations. The p-ethyl-phenol and equol concentrations that we detected in plasma of cows fed soybean (1.6 ± 0.31 µM and 1.2 ± 0.28 µM, respectively) were more than a thousand times greater than the concentrations of endogenous E2 (1–10 nM) (50). These high concentrations may compensate for the much weaker affinity of phytoestrogens for estrogen receptors (47). It has been previously shown that the concentrations of phytoestrogens in plasma of pregnant women consuming soybeans are over 1000 times higher than E2 concentrations and 10,000–100,000 higher than E2 concentrations during the menstrual cycle (1, 17, 24). The phytoestrogen concentrations used in the present in vitro study (10 nM) were lower than the concentrations of conjugated phytoestrogens found in the blood plasma (1.6 ± 0.31 µM and 1.2 ± 0.28 µM). About 5%–0% of all phytoestrogens in bovine plasma are in the free form (34, 40). The concentrations of free phytoestrogens and their metabolites were below the detection limit of our HPLC system. Thus, the concentrations of phytoestrogens and their metabolites that we used in the present in vitro study were based on the free, unconjugated daidzein (0.2–0.4 nM) and equol (4–8 nM) found by Lundh et al. (34, 35) in plasma of cows fed with moderately estrogenic silage. In our in vitro experiments, phytoestrogen metabolites (equol and p-ethyl-phenol) turned out to be much more potent disruptors than the original phytoestrogens themselves. The stronger effects of the metabolites may be due to their higher affinities for estrogen receptors than original phytoestrogens. This hypothesis is supported by findings of other authors (reviewed in 2, 47) who showed that phytoestrogen metabolites are about 100%–150% more active than environmental estrogens.

Phytoestrogens derived from soybean and their metabolites stimulated PGF2{alpha} and PGE2 production in the cultured bovine endometrium at different stages of the estrous cycle. However, the strongest effects of phytoestrogen metabolites (p-ethyl-phenol and equol) on PGF2{alpha} secretion were observed in the phases of the estrous cycle (early II and midluteal; Table 3Go). Prostaglandin E2 and PGF2{alpha} are crucial for proper development and maintenance of the CL. The maintenance of CL and P4 production is regulated by several luteotropic factors, including PGE2 (51). Stimulation of PGF2{alpha} production disturbs the ratio PGE2 to PGF2{alpha} Proper PGF2{alpha} to PGE2 ratio is important for the maternal recognition of pregnancy, for maintaining the function of CL, and embryo implantation and development (30, 52). A strong stimulation of PGF2{alpha} production by phytoestrogens or their metabolites can lead to a disturbance (i.e., an increase) of this ratio that may interfere with early embryo development and implantation. During embryo development and implantation the PGF2{alpha} to PGE2 ratio should decrease. This relaxes the blood vessels and increases blood flow in the uterus, which prepares it for embryo implantation (31). The decreased PGF2{alpha} to PGE2 ratio also stimulates P4 synthesis (53). In our study, phytoestrogens and their metabolites greatly increased PGF2{alpha} production and moderately but significantly increased PGE2 production. These changes may interfere with embryo implantation in the uterus. Because PGF2{alpha} has a direct and negative effect on bovine embryo development in vitro (54), the strong stimulation of PGF2{alpha} production compared with PGE2 production that we observed (6–7 times greater; Table 3Go) may be one of the reasons for the early embryo mortality or abortion.

Phytoestrogens and their metabolites also strongly stimulated PGF2{alpha} output from the bovine endometrium at the late luteal and follicular phases of the estrous cycle. Stimulation of PGF2{alpha} secretion by estrogenic-like substances during luteolysis (i.e., during the late luteal phase and follicular phase) may accelerate the positive feedback loop between PGF2{alpha} and other regulators of luteolysis, such as oxytocin (OT) (26, 30, 52, 55). Estradiol-17ß increases OT- stimulated PGF2{alpha} production in cultured bovine endometrial cells (32) as well as amplifies the stimulatory effect of OT on endometrial PGF2{alpha} synthesis (56). Additionally, gonadal steroids upregulate OT gene expression in the hypothalamus and upregulate OT receptors in the uterus; thus, they can alter the frequency of the central OT pulse generator, leading to the pulsatile PGF2{alpha} output from the endometrium during luteolysis in ruminants (30, 57). Therefore, if phytoestrogens and their metabolites act like endogenous estrogens, especially in nonpregnant animals, at the time of luteolysis and ovulation, they may amplify the mechanisms that return the cow to cyclicity after labor.

In view of the structural and functional similarity of phytoestrogens, their metabolites, and endogenous estrogens, we presume that plant-derived estrogens that are present in the plasma of cows fed soybean modulate PG synthesis in the bovine endometrium at the enzyme level. Thus, phytoestrogens and their metabolites, like endogenous estrogens, may influence the activity and expression of key enzymes taking part in the PG synthesis (26, 58): cyclooxygenase-2 (COX-2) (32, 59), PG synthases (60), and even PGE-9-keto-reductase, which converts PGE2 to PGF2{alpha} (61, 62). Our previous in vitro study (63) revealed that phytoestrogens and their metabolites affected the expression of COX-2 and PGE synthase in cultured bovine stromal and epithelial cells of the endometrium. Equol and p-ethyl-phenol strongly stimulated PGF synthase expression at the protein level in the epithelial cells (63), which are the main source of luteolytic PGF2{alpha} production in the bovine endometrium (32, 62). Thus, phytoestrogens may reduce the PGE2 to PGF2{alpha} ratio during the estrous cycle and pregnancy. Reduction of the PGE2 to PGF2{alpha} ratio would modulate the expression of key enzymes taking part in PG synthesis in the bovine endometrium.

The presented data provide a possible explanation of how soybean-derived phytoestrogens and their metabolites act as endocrine disruptors, leading to disruption of the reproductive processes and to temporal infertility of cows. Phytoestrogens and their active metabolites disrupt the ratio PGE2 to PGF2{alpha}, which leads to the nonphysiological production of luteolytic agent in cattle during the estrous cycle and pregnancy. Therefore, phytoestrogen-dependent inhibition of the PGE2 to PGF2{alpha} ratio might be the reason for the early embryo mortality that occurs with cows fed a high-soy diet.


   Acknowledgments
 
We thank Dr. Seiji Ito of Kansai Medical University, Osaka, Japan, for PGE2 antiserum; and Dainippon Pharmaceutical Co., Ltd., Osaka, Japan, for recombinant human TNF{alpha} (HF-13). We thank CENTROWET, Olsztyn, Poland, for the gifts of Crestar and Union Nord-Ouest Genetique Bosc-Berenger, France, for the gift of bull semen used in this study. We also thank Mrs. Hanna Kostuch and Mr. Jerzy Kostuch from the Animal Farm "Farmer" in Zalesie, as well as Krystyna Kasinska and Zenon Kasinski from the Animal Farm P.R.H. "Watkowice" in Watkowice for the present in vivo examination. The authors are indebted to Dr. James Raymond for critical review of this manuscript and English correction.


   Footnotes
 
This research was supported by Grant-in Aid for Scientific Research from the Polish Ministry of Scientific Research and Information Technology (grant KBN 5P06K00321). Parts of these data were presented at the joint meeting of the Section of Fertility and Infertility of Polish Gynecology Society and the Society for Biology of Reproduction in Bialowieza, Poland, June 10 to June 12, 2004. In vitro experiments (nos. 2 and 3) are part of the Ph.D. thesis of Izabela Woclawek-Potocka.

Received for publication June 15, 2004. Accepted for publication December 2, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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