C. F. Rosenkrans, Jr, B. C. Paria, D. L. Davis and G. Millikenof estradiol, catechol estrogen and ascorbic acid

In vitro synthesis of prostaglandin E and F2 alpha by pig endometrium in the presence

1990, 68:435-443.J ANIM SCI 

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IN VITRO SYNTHESIS OF PROSTAGLANDIN E AND F2a BY PIG ENDOMETRIUM IN THE PRESENCE OF ESTRADIOL, CATECHOL

ESTROGEN AND ASCORBIC ACID"*

C. F. Rosenkrans, Jr.3, B. C. F'aria3, D. L. Davi? and G. Milliken'

Kansas State University, Manhattan 66506

ABSTRACT

These experiments were undertaken to determine the potential for estradiol-17P (Ed, 2-hydroxyestradiol-17p (2-OH-Ez) and 4-hydroxyestradiol-17P (4-OH-Ez) to regulate prosta- glandin (PG) E and F2a synthesis by pig endometrium. Endometrium was collected from pigs on d 10 of pregnancy and incubated (15 to 20 mglwell) for three 2-h periods in 2 ml of medium in 24-well culture plates. At the end of each period. the medium was removed and frozen. Later, media were thawed and assayed for PGE and PGFza. During Periods 2 and 3, the medium contained 0,25,50,100 or 150 pA4 2-OH-E2 (Exp. 1); 0,25 or 50 pM 4-0H-Ez (Exp. 2); or 0, 25 or 50 phf E2 (Exp. 3). Each experiment was a factorial with 2-OH-Ez, 4-OH-E2 or E2 as one main effect and 0 or 1 mM ascorbate as a second main effect. Ascorbate decreased (P < .01) PGE and PGFza release in all experiments. Two-hydroxyestradiol-17p decreased (P < .Ol) PGE and K F z a release into the medium during Periods 2 and 3 in a dose-dependent manner (Exp. 1). In Exp. 2, 4-OH-Ez decreased (P < .07) endometrial release of PGE and PGF2u in Periods 2 and 3 and increased (P < .01) the PGE:PGFn in Period 3. In Exp. 3, Ez decreased release of PGE during Period 3 and PGFza release during Period 2. The PGE:FGF2a was not altered by Ez. Non-incubated endometrium contajned only small amounts of PGE and PGFza (< 30% of that released during 2 h of culture), similar to the content of endometrium after incubation. Therefore, PG released in vitro represent de novo synthesis of PG. Catechol estrogens, Ez and ascorbate inhibited PG synthesis by pig endometrium. Therefore, those compounds may mediate endometrial PG synthesis during establishment of pregnancy in pigs. (Key Words: Estrogens, Catechol Estrogens, Endometrium, Pregnancy, Pigs.)

J. h i m Sci. 1990.68:435-443

Introduction

The ability of pig blasmcysts to synthesize estrogen was reported initially by Perry et al. (1973) and more recently estradiol-2/4-hydroxy-

'This work was upp ported by USDA grant 85CRCR- 1-1872 (to D.L.D.). Published as contribution 89-2424 of the Kansas Agric. Exp. Sta

zWe thank C. Chalaaborty for HPLC analysis of cate- chol estrogens, N. R. Mason, Eli Lilly and Co., Indianap- olis, IN for providing the prostaglandin Ez antiserum. K. Richardson, M. Krause and J. CaTpenter for technica as- sistance and V. Brock and G. Weir for typing the manu- scri t ?De$ of Anin Sci. and Ind. 4Address requests for reprints to this author.

Received February 27, 1989. Accepted June 13,1989.

5Dept. of stat.

lase (E-2/4-H) (Mondschein et al., 1985; Chakraborty et al., 1989) and 15a-hydroxylase (Chakraborty et al., 1988) activities have been observed in pig blastocysts. The E-2/4-H con- verts estradiol (Ez) to catechol estrogens (2-OH- E2 and 4-OH-Ed. Catechol estrogens stimulate and alter the relative amounts of prostaglandins (PG) synthesized by the rat uterus (Kelly and Abel, 1980). human and rabbit endometrial cells (Kelly et al., 1983; Pakrasi and Dey, 1983) and rabbit blastocysts (Pakrasi and Dey, 1983).

Prostaglandins E and F increase in the uterine lumen and in blastocysts of pigs after 10 d of pregnancy (Giesert et al., 1982; Davis et al., 1983) and may participate in stimulation of in- creased uterine blood flow (Ford and Christen- son, 1979), vascular permeability (Keys et al.,

435

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436 ROSENKRANS ET AL.

1986) and attachment to uterine epithelium (Dan- Der, 1985). Pig embryos exhibit a surge in estro- gen and catechol estrogen synthesis between d 11 and 14 (Mondschein et al., 1985)and catechol estrogens are synthesized by endomemal tissue (Van Orden et al., 1986). Therefore, we investi- gated the effects of E2,2-OH-Ez,4-OH-Ez and ascorbic acid on synthesis of PGE and PGF2a by pig endometrium in vitro. We included ascorbic acid in these experiments because it is used com- monly to protect catechol estrogens from autox- idation (Gelbke and Knuppen, 1972) and because it is found in the uterine lumen of pigs during early pregnancy (zavy et al., 1982).

Materials and Methods

Endometrium was collected surgically from Yorkshire x Chester White x Hampshire sows on d 10 of pregnancy. Embryos (1 to 5 mm in diam- eter) were flushed from the uterus using phos- phate-buffered saline (PBS)6 as described previously (Chakraborty et al., 1988). Immdi- ately after embryos were collected, infusion can- n u l a ~ ~ were inserted into adjacent branches of the uterine artery, and PBS was infused to flush most of the blood from the vascular system of a short segment of uterus because red blood cells might convert catechol estrogens to their methoxy de- rivatives (Bates et al., 1977). The uterus was opened longitudinally on the antimesometrial surface, and endomemal strips were removed. The uterine and abdominal incisions were closed using standard procedures and pigs were allowed to recover. Fine scissors were used to cut the endometrium into 1- to 2-mm cubes, and 15 to 20 mg of endometrium were incubated per well in 24-well cultureplates6. Each well contained 2 ml of a modified Krebs-Ringer bicarbonate medium (mKRB; Davis and Day, 1978) supplemented with the vitamins and amino acids (essential and nonessential) in Minimum Essential Medium6. The medium did not contain phenol red, bovine serum albumin or antibiotics. Incubation mix- tures were rocked gently (two oscillations/min) with temperature (39'C) and atmosphere (95% air and 5% C02) maintained in a humidified chamber*.

6clBC0, Grand Island, NY. '21-gauge, Terumo Corp., lokyo, Japan. sBillups-Rothenburg Inc., Del Mar, CA. gSteraloids, Wilton, NH.

'?asteur Inst., Mames La Coquett, France.

Endometrium was collected from 10 sows, 3 or 4 sows for each experiment. Incubations were conducted for three 2-h periods. During the first period, no treatment was imposed, and the endo- metrium was allowed to equilibrate with the in vitro environment. At thebeginning of the second and third periods, the medium was replaced with fresh medium containing 0 or 1 mM ascorbic acid and the appropriate catechol estrogen or E? treat- ment in 10 pl of ethanol. One incubation treat- ment received the same treatment in Periods 2 and 3. The ethanol also contained 1 mM ascorbic acid (final contribution of ascorbic acid to the culture medium was .0005 M). Purity of 2-OH- Ez9 and 4-OH-Ez9 was greater than 95% as indi- cated by HPLC and electrochemical detection (Chakmborty et al., 1988).

Effects of 2-OH-E2 (0, 25, 50, 100 and 150

and E2 (0,25 and 50 pM; Exp. 3) were tested in factorial experiments with 0 or 1 mM ascorbic acid. Following 6 h of incubation, 20 pg of indo- methacin were added to each well, and the endo- metrium and media were separated by cenmfugation (2,000 x g for 15 min at 5'C). Endometrial tissue and media were stored sepa- rately at -60°C until they were assayed for PGE and PGF2a.

Prostaglandins were quantified by RIA with- out chromatography, using methods similar to those described by Lewis et al. (1978) for the assay of PGE. Culture media were assayed with- out extraction. Endometrial tissues were homog- enized and sonicated then acidified @H c 4) with 1 N hydrochloric acid and extracted three times with ethyl acetate (efficiency > 90%). The PGF2a antiserum" (diluted 1: 13) cross-reacted less than 1% with PGEl, F'GE2, PGFla, FGA2, 13.14-dihydro- 15-keto-PGE2, 1 3- 14 -di hydro- 15-keto-PGFza and arachidonic acid. When 50, 100, 250 and 500 pg of PGF2a were added to mKRB, the immunorecovery of added PGF2a was 112% and the correlation between added and recovered PGF2a was .96. Increasing volumes (0 to 200 @) of medium from incubated endome- trium or extracts of endometrium produced bind- ing curves that paralleled the standard curves. The intra- and inter-assay coefficients of varia- tion were 13.8% and 20.8%. respectively.

The PGE antiserum (kindly donated by N. R. Mason, Eli Lilly, Indianapolis, IN) was diluted to a final concentration of 1:20,000 and cross-re- acted 17% with FGEl but less than 5% with other PG (Lewis et al., 1978). Therefore, results are reported as immunoreactive PGE. Adding 5, 10,

w; EXP. l), 4-OH-Ez (0.25 and 50 JLM; EXP. 2)

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PROSTAGLANDIN SYNTHESIS BY PIG ENDOMETRIUM 437

TABLE 1. PROSTAGLANDIN (PG) CONTENT OF PIG ENDOMETRIUM AT RECOVERY

OR CATECHOL ESTRADIOL AND AFTER 6-IIOUR INCUBATION IN MEDIA CONTAINING ESTRADIOL

K F 2 a , P g I d endc-

Palma No. of endo-

Item explants metrium metrium

Exp. 1 2- hydroxyestradiol-17P, pM

Not incubated 0

25 50

100 150 Pooled SEM

Exp. 2 4-hydroxyestradiol-l7~, pM

Not incubated 0

25 50 Pooled SEM

Exp. 3 Estradiol-17P. pM

Not incubated 0

25 50 Pooled SEM

3 6 6 6 6 6

3 6 6 6

3 8 8 8

1.2 (3)a 2.1 (3) 2.4 (5) 2.0 (6) 2.6 (3) 2.1 (3)

.5

.9 (3) 1.7 (6) 1.9 (6) 1.1 (4) .5

1.2 (2) 1.1 (8) .8 (8) .8 (8) .2

aNo. of explants with detectable PG. Amounts 2.8 pg/mg would have been detected by the assays in 15 mg of endometrium and some explants were p l e d such that 15 to 70 rng of endometrial homogenate were included in each assay tube..

25,50 and 100 pg of PGE2 to mKRB yielded an 80% immunorecovery of added PGE2 and the correlation between added and recovered PGE2 was .99. Increasing volumes (0 to 200 pl) of medium from incubated endometrium or endo- metrial extracts produced binding curves that paralleled the standard curves. The intra- and inter-assay coefficients of variation were 10.3% and 11.8%, respectively. Sensitivity of both as- says was 1.25 pglassay tube.

Prostaglandins are exprcssed as pg/mg of en- dometrium (wet weight) incubated. The PGE:PGFza (PG-ratio) also was calculated. Sta- tistical analyses were conducted using the Gen- eral Linear Models procedure (SAS, 1982). Data were analyzed as factorials with estrogen and ascorbic acid treatments as main effects and sows as blocks. For each experiment, periods two and three were analyzed individually and Period-1 PG release was included as a covariate. Linear

and quadratic responses to E2 and catechol estro- gens were examined using CONTRAST and ESTIMATE statements.

Results

The PGE and PGF2a contents in unincubated endometrium and in endometrium after incuba- tion (Table 1) were similar, were not affected by treatments and were less than 30% of that re- leased in 2 h by untreated endometrium. Release of PGE and PGF2a (Figures 1 to 3) during Peri- ods 2 and 3 was correlated (r > .48; P c .01) with their respective release during Period 1. There- fore, PG release during Period 1 was included as a covariate in statistical models and its inclusion decreased the means square errors.

Experiment 1. Medium from Period 1 con- tained 22rt 2 and 25 k 1.7 pg/mg of endometrium of FGE and PGFza, respectively, and the PG- ratio was .9 k .05 for Period-1 medium. Both

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438 R 0 S E ” S ET A L

2 - HYDROXYESTRADI OL m OpM 0 25pM 50pM = 100pM IXI 150pM

25 25 w- 2

4 2 15

o\ 5 E g 5

A 0 mM Ascorbate 1 mM Ascorbate 20 2 . $ 2 0

z 3 15

0 3 4 10 I- * l o m E

0 0

1 mM Ascorbate 1.5 1.5

8 c\l 0 mM Ascorbate LL.

1 .o 2 1.0 .. W c3

.5 a .5

.o .O 2 3 2 3

INCUBATION PERIOD Figure 1. Release of prostaglandin (PG)E (A) a n d F p (B) and theirratio (C) during incubation in medium containing

2-hydroxyestradiol (2-OH-E2) with or without ascorbic acid. Each bar is X ?c SEM of media from 12 incubations (four replicates in each of three sows). (A) Ascorbate and 2-OH-Ez (P c .01) decreased PGE release. (B) Ascorbate and 2-OH-Ez decreased (P < .01) PGFza release. (C) Ascorbate decreased (P < .05) PGE:PGFp.

2-OH-Ez and ascorbate decreased (P < .01) FGE and PGFza release into the medium during Pen- ods 2 and 3 (Figure 1). Quadratic (P c .OS) effects described the decreases in PGE and PGF2a re- lease attributable to 2-OH-Ez during Period 2 and

FGFza release in Period 3. During Period 2, PGF2a release was affected by an interaction between 2-OH-Ez and ascorbic acid. That inter- action was due to a linear (P c .OS) decrease in the release of PGFza with dose of 2-OH-E2 in the

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PROSTAGLANDIN SYNTHESIS BY PIG ENDOMETRIUM

4- HYDROXY ESTRAD IO L m OpM 0 25pM 50pM

20

A ? 0 mM Ascorbate T

439

1 mM Ascorbate

T 10

5

0

1 mM Ascorbote

0 mM Ascorbate

2 3 2 3 IN CUBATI ON P ERlO D

Figure 2. Release of prostaglandin (F'G)E (A) and Fza (B) and their ratio (C) during incubation in medium containing 4-hydroxyestradid (4-OH-Ez) with or without ascorbic acid Each bar is E f SEM of media from 12 incubations (four replicates in each of three sows). (A) Ascorbate (P < .M) and 4-OH-Ez (P < .M) decreased the release of PGE. (B) Release of P G F s was decreased by ascorbate (P < .001) and 4-OH-Ez (P < .01). (C) the PGE:PGFp was increased (P < .Ol) in Period-3 media by 4-OH-Ez.

presence of 1 mM ascorbic acid. In the absence of ascorbic acid, the initial decline in PGF2a release was more rapid and best described by a decay curve from 0 to 50 pA4 2-OH-E2 and a linear decrease from 50 to 150 pM.

Experiment 2. Period-1 medium contained 19 k 2.4 and 18 k 2.4 pg/mg PGE and PGFZa, respectively, and the PG-ratio was 1.2 k .08.

Release of PGF2a during Periods 2 and 3 de- creased (P c .01) linearly with dose of 4-OH-Ez (Figure 2). During Period 2, the release of PGE was decreased linearly (P c .OOl) by 4-OH-Ez, but in Period 3 the response was quadratic (P c .07). Ascorbic acid decreased release of FGE (P c .06) and PGFza (P c .OOl) during both periods. The PG-ratio for Period 2 was not af-

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440 ROSENKRANS ET AL.

ESTRADIOL- 1 78

INCUBATION PERIOD Figure 3. Release of prostaglandin (PG) E (A) and P G F s (B) and their ratio (C) during incubation in medium

containing eswadiol-l7a @d with or without ascorbic acid. Each bar is jT k SEM of media f?om 24 incubations (six replicates in each of four sows). (A) During Period 3 the release of F’GE was decreased by E2 ( P < .05) and ascorbate (P = .W) and by an E2 x ascorbate interaction (P = .07). (B) Ascorbic acid decreased (P < .Os> =lease of FGF2a during Period 3. (C) Ratio of the PG was not affected (P > .lo) by the treatments.

fected (P > .20) by treatment, but during Period 3 it was increased (P < .01) by 4-OH-Ez.

Experiment 3. During Period 1, the endome- trial tissues released 18 k 1.6 and 23 k 1.9 pg/mg of PGE and PGFza, respectively. and the PG- ratio was .9 k .08. Release of FGE during Period 2 was not affected (P > .40) by E2 or ascorbate,

but there was a tendency (P = .07) for an ascorb- ate x E2 interaction, indicated by a small depres- sion in PGE-release in the presence, but not in the absence, of ascorbate (Figure 3). Release of PGE during Period 3 was decreased linearly (P < .05) by E2 and also tended (P = .07) to be suppressed by ascorbate. Ascorbic acid decreased (P < .05)

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PROSTAGLANDIN SYNTHESIS BY PIG ENDOMETRIUM 441

FGF2a release during period 3, but E2 did not affect (P > .14) the release of PGF2a during Pe- riods 2 or 3. The PG-ratio was not affected by either ascorbic acid or E2.

Discussion

Both primary and catechol E2 decreased PG synthesis by d-10 pig endometrium in vitro and 4-OH-E2 increased the PGE:PGF2a. That shift in PG-ratio might be important for pregnancy estab- lishment. Receptors for PGEz are present in the pig uterus in early pregnancy (Kennedy et al., 1986). Prostaglandins of the E-series cause dila- tion of the uterine vascular bed in ewes (Resnik and Brink, 1978; Still and Greiss, 1978) and uterine blood flow increases on d 12 of pregnancy in pigs (Ford and Christenson, 1979) coincident with peaks in aromatase and catechol estrogen synthesis by pig blastocysts (Mondschein et al., 1985). Therefore, embryonic estrogens might alter PG-ratios to enhance uterine blood flow. Increased capillary permeability in the endome- trium adjacent to d-13 pig blastocysts also has been reported (Keys et al., 1986) and PGEz is more effective than PGFza for increasing capil- lary permeability in the rabbit uterus (Hoffman et al., 1977). Catechol estrogens also might act in- dependently of PG, through effects on potential- sensitive calcium channels, to increase uterine blood flow (Stice et al., 1987).

Peroxidase-mediated synthesis of catechol es- trogens by the pig endometrium produces rela- tively large amounts of 4-OH-E2 (Van Orden et al., 1986). Conversion of blastocyst-synthesized estrogens to 4-hydroxyestrogens could be a mechanism for altering the PGE:PGFza in the gravid uterus. In contrast, a cytochrome-P-450 mediated monooxygenase in pig blastocysts me- tabolizes E2 primarily to 2-OH-Ez (Mondshein et al., 1985) and 2-OH-E2 shifts the PGE:PGFza synthetic profile in blastocysts in favor of FGE (Rosenkrans et al., 1989). Therefore, differential metabolism of E2 to 4- vs 2-OH-E2 in the endo- metrium vs blastocyst may be relevant to FGE:PGF ratios greater than one in the uterine flushings collected on d 12 to 14 of pregnancy (F'aria and Rosenkrans, 1988). The mechanism by which 4-OH-Ez shifts the F'G-ratio in favor of PGE might result from dilferential effects on different components of the endometrium. Re- cent studies (Zhang et al., 1989) show that iso- lated uterine glands are more active in PGF than PGE synthesis and stromal cells synthesize more PGE than PGF. Fortier et al. (1988) also reported

differential PG-ratios for epithelial and stromal components of bovine endometrium. Prostaglan- din synthesis in uterine glands is inhibited by 4-OH-E2, but 4-OH-Ez stimulates PG synthesis in stromal cells (Zhang et al., 1989). Therefore, the net effect of catechol estrogens on endome- trial PG synthesis would be an increased PGE:PGF2a, which may be important for preg- nancy establishment.

In contrast to our results, Pakrasi and Dey (1983) reported that 2-OH-E2 and 4-0H-Ez in- creased production of FGE-A and PGF by rabbit endometrial cells and E2 stimulated PGE-A pro- duction. Kelly and Abel(l980) reported that both E2 and 2-OH-E2 increased PGE2 and PGFza re- lease by homogenates of rat uteri. We (Zhang et al., 1989) observed that 4-OH-Ez stimulated PG synthesis in stromal cells but inhibited PG syn- thesis in cultures of uterine glands of pigs. There- fore, both species and cell type affect the response of the uterus to catechol estrogens.

Concentrations of catechol estrogens used in our experiments were similar to those used by other investigators (Kelly and Abel, 1980; Pakrasi and Dey, 1983). In preliminary studies, pig endometrium was treated with lower concen- trations (.025 to 7.5 CLM) of catechol estrogens and effects on PG release were not detected (Rosenkrans, 1988). Others (Weisz, 1983) have reasoned that relatively large concentrations are required to elicit effects because catechol estro- gens are rapidly metabolized in vitro (Kelly et al., 1983). Mondschein et al. (1985) reported that mmatase and estrogen-2/4-hydroxylase activi- ties peak on d 12 and 13 in pig blastocysts, but the concentrations of estrogens and catechol es- trogens in the endometrium immediately adja- cent to the embryo are not known. In the pig endometrium, conversion of blastocyst-produced primary estrogens to catechol estrogens might result in relatively large concentrations of cate- chol estrogens within endometrial cells.

We utilized endometrium collected on d 10 of pregnancy, when pig embryos produce estrogen and catechol estrogens (Dey et al., 1984; Mondschein et al., 1985) but the surge in both synthetic activities has not yet begun. Therefore, it is likely that supplemental E2 or catechol estro- gens would advance endometrial responses. Tis- sue already exposed to surges of estrogens might be less responsive. We evaluated the effects of catechol estrogens on endometrium from preg- nant pigs because it is possible that it would respond differently from endometrium from cy- clic pigs. Perhaps endometrium from pregnant

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442 ROSLNKRANS ET A L

pigs from d 12 or 13 would respond differently from d- 10 endometrium from pregnant pigs. We have not evaluated those possibilities, but the effects of catechol estrogens on enzymatically dissociated pig endometrium, collected from d 10 to 19 of pregnancy, are consistent with the results reported here (Zhang et al., 1989 and unpublished data). Therefore, we believe our present results may be representative of the response of endome- trium to E2 and catechol estrogens during early pregnancy in pigs.

Ascorbic acid decreased synthesis of PG by about 20%. This agrees with results of Kelly and Abel(1980), who found that ascorbate (1.0 mM) reduced the stimulatory effects of 2-OH-E2 on PG release. However, those authors also reported that addition of ascorbate in the absence of cate- chol estrogens stimulated FG synthesis. Reasons for that contradiction are not known.

The mechanism by which E2 and catechol estradiols decrease synthesis of PG in the pig endometrium is not known. Stice et al. (1987) reported that 4-OH-Ez blocks calcium uptake via potential-sensitive channels in pig uterine arter- ies. Because our expbnts included vascular tis- sues and because the calcium ionophore A23 187 increased PG synthesis in cndometrial explants (Rosenkrans, 1988), perhaps E2 and the catechol estrogens decreased PG generation by blocking calcium uptake.

Others have suggested that catechol estrogens stimulate PG synthesis by serving as metal che- lators (Kelly et al., 1983) or as co-substrates for PGH synthesis (Degen et al., 1987). Catechol estrogens also function as antioxidants and are more effective than a-tocopherol for preventing phospholipid peroxidation of membranes (Nakano et al., 1987). Antioxidants can affect many aspects of PG synthesis (Carpenter, 1981) and often inhibit PG generation by removing intermediate radicals that are essential for the cyclooxygenase mechanism (Hemler and Lands, 1980). Therefore, the antioxidant properties of catechol estrogens may be involved in their ef- fects on PG synthesis.

Implications

The results of our experiments indicate that estradiol and catechol estradiols may be involved in the control of endometrial synthesis of prosta- glandins in the pig. Their short-term effects in our studies were inhibitory, and 4-hydroxyestradiol altered the prostaglandin-ratio in favor of prosta- glandin E. Prostaglandins are implicated in preg-

nancy establishment. Therefore, our data suggest a mechanism by which estrogens could modify PG synthesis to exert efforts on pregnancy estab lishment in pigs.

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