Molecular and Cellular Endocrinology, 69 (1990) 25-32 Elsevier Scientific Publishers Ireland. Ltd.
MOLCEL
25
02221
Catechol estrogen formation in the mouse uterus and its role in implantation Bibhash C. Paria, Chandan
Chakraborty
and Sudhansu
K. Dey
Department of Obstetrics and Gynecology and Physiology, University of Kansas Medical Center, Ralph L. Smith Research Center, Kansas City, KS 66103, U.S.A. (Received
Key words: Catechol
estrogen;
28 July 1989; accepted
Estradiol-2/4-hydroxylase;
31 October
Estradiol-4-hydroxylase;
1989)
Prostaglandin;
Peri-implantation;
(Mouse uterus)
Summary Estradiol-2/4-hydroxylase (E-2/4-H) activity was determined in the mouse uterus during early pregnancy as well as in ovarian steroid hormone-treated ovariectomized uterus. Under the assay conditions used, E-4-H was the predominant catechol estrogen-forming monooxygenase enzyme. The inhibition of E-4-H activity by SKF-525A, metyrapone and a-naphthoflavone suggested involvement of cytochrome P,,,-dependent monooxygenases. A haloestrogen, 2-fluoroestradiol (2-FL-E,), also inhibited this activity. During the peri-implantation period, no change in uterine E-4-H activity was noted on the morning of days 2 through 5, but the activity significantly (P < 0.01) increased in the afternoon of day 4 of pregnancy, A single injection of estradiol-17fl (E,, 100 rig/moose)) to ovariectomized mice significantly (P -=z 0.01) elevated the level of E-4-H activity at 24 h as did injections of progesterone (P4, 2 mg/mouse) for 2 days. When 2 days of P4 (2 mg/mouse) treatment was combined with a single injection of E, (20 ng/mouse), E-4-H activity increased 1.3-fold (P < 0.05) by 24 h above that of P4 treatment alone. Dexamethasone (200 pg/mouse) and cholesterol (2 mg/mouse) treatment for 2 days had no effect on E-4-H activity. Thus, the stimulatory effect of P4 and E, on E-4-H activity appeared to be specific. The increased activity of uterine E-4-H prior to implantation on day 4 evening and the modulation of its activity by Ps and/or E, suggest an involvement of 4-hydroxyestradiol in embryo implantation. In delayed implanting mice, while E, as low as 10 rig/moose induced implantation in all animals, a dose of 2-FL-E, as high as 100 rig/moose failed to induce implantation in all animals. Furthermore, administration of 2-FL-E, (50 rig/moose)) just before E, (10 rig/moose)) injection completely blocked estradiol-inducible implantation. Interestingly, administration of different doses of prostaglandin E, (PGE,, 2-5 pg/mouse) when given in combination with 2-FL-E, as low as 25 rig/moose induced implantation in most of the animals. These results suggest that at least some of the important actions of estrogen in implantation are mediated via formation of catechol estrogens and PGs.
Introduction Address for correspondence: S.K. Dey, Ph.D., Department of Obstetrics and Gynecology and Physiology, University of Kansas Medical Center, Ralph L. Smith Research Center, 39th and Rainbow Boulevard, Kansas City, KS 66103, U.S.A. 0303-7207/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
In the mouse or rat, requirement for initiation Ltd.
estrogen is an absolute of embryo implantation
26
in a progesterone (P4)-primed uterus (Psychoyos, 1973; Huet and Dey, 1987). However, the mechanism by which estrogen initiates this process is not clearly understood. Recently, it has been proposed that estrogen action in implantation is mediated via local formation of catechol estrogens (CEs) in the target tissue (Hoversland et al., 1982; Kantor et al., 1985; Mondschein et al., 1985; Dey et al., 1986). CEs are the major aromatic hydroxylation products of phenolic estrogens (Fishman, 1983). The formation of CEs occurs primarily in the microsomes and is catalyzed by estradiol-2/4-hydroxylase (E-2/4-H) which is an NADPH-dependent cytochrome PdsO monooxygenase-associated enzyme (Paul et al., 1977). There are also indications that more than one form of P450s may mediate the formation of CEs (Lang and Nebert, 1981) or they could be formed by peroxidase system (Levin et al., 1987). The formation of CEs has been demonstrated to occur in a wide variety of normal and neoplastic tissues (Poth et al., 1983). CEs can bind to estrogen receptors and have biological effects as estrogen agonists or antagonists (MacLusky et al., 1981; Schneider et al., 1984). However, because of extremely short half-life and rapid clearance rate (Gelbke et al., 1977; MacLusky et al., 1981; Schneider et al., 1984) CEs are not likely to function as circulating hormones. Therefore, an attractive hypothesis is that they may act as local mediators of estrogen action in estrogen-responsive target tissues. A surge in E-2/4-H activity was noted in the pig blastocyst on days 12 and 13 of pregnancy that corresponds to the stage of pregnancy recognition (Mondschein et al., 1985). Furthermore, circumstantial evidence suggests that CEs are important for implantation in the mouse and rat (Hoversland et al., 1982; Kantor et al., 1985). However, no information is available whether the uterus and/or the embryo in these species have the capacity to form CEs during the peri-implantation period. Therefore, the temporal pattern of E-2/4-H activity was studied in the mouse uterus during this period. Since the process of implantation is primarily dependent upon an interaction between P4 and E, (Psychoyos, 1973) the effects of these steroids on uterine E-2/4-H activity were also investigated in the ovariectomized mice.
Prostaglandins (PGs) are considered to be involved in the process of implantation (Kennedy, 1977), and CEs are more potent than phenolic estrogens in stimulating PG synthesis (Kelly and Abel, 1981; Pakrasi et al., 1983). Therefore, it could be further postulated that inhibition of formation of CEs and/or PGs will interfere with implantation. Thus, effects of 2-fluoroestradiol (2FL-E,), an inhibitor of E-2/4-H, on implantation were also investigated. Materials and methods Chemicals 2-Hydroxyestradiol (2-OH-E,) and 4-hydroxyestradiol (4-OH-E,) were purchased from Steraloids (Wilton, NH, U.S.A.). [4-‘4C]E, was obtained from New England Nuclear (Boston, MA, U.S.A.). HPLC-grade methanol and sodium acetate were obtained from Fisher Scientific (St. Louis, MO, U.S.A.). Glacial acetic acid (HPLCgrade) was purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, U.S.A.). Estradiol-17/I (E2), progesterone (P4), reduced nicotinamide adenine dinucleotide phosphate (NADPH), DL-dithiothreitol (DTT), Hepes, Tris, ascorbic acid, glycerol, E-64 (trans-epoxysuccinyl-L-leucylamido-4-guanidino-butane), metyrapone (2-methyl-1,2-di-3pyridyl-propanone) and a-naphthoflavone (7,8benzoflavone) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). SKF-525A was obtained from Smith-Kline and French Laboratories (Philadelphia, PA, U.S.A.). 2-FL-E, was prepared, characterized and generously provided by Dr. J.G. Liehr (University of Texas Health Science Center, Galveston, TX). Animals CD-1 female mice (48 days old, 20-25 g, Charles River, NJ, U.S.A.) were housed in temperature(25” C) and light-controlled (14:lO h) quarters and had free access to food and water. Female mice were mated with fertile males of the same strain. The morning of finding vaginal plug was designated as day 1 of pregnancy. Experiment I To determine activity. mice
temporal pattern of uterine E-4-H were killed in the morning
27
(08.30-09.00 h) of days 1 through 5, except one group which was killed in the afternoon (15.0015.30 h) of day 4. The reproductive tracts were flushed for embryos to confirm pregnancy. The uteri were cleaned of adhering fat and mesentery, lightly blotted, weighed and kept in ice-cold TEGD buffer (see below) for E-2/4-H assay.
Experiment 2 To explore the effect of steroids on E-2/4-H activity, mice were ovariectomized regardless of their stages of the estrous cycle. After 5 days of rest, animals were divided into eight groups. Group 1 served as controls, i.e., they received only the vehicle (0.1 ml sesame seed oil). While mice in group 2 were killed 4 h after a single injection of E, (100 ng/O.l ml sesame seed oil/mouse, s.c.), those in group 3 were killed 24 h later. Mice in group 4 received injections of P4 (2 mg/O.l ml oil/mouse/day, s.c.) for 2 days and killed 24 h later. Mice in groups 5 and 6 were injected with the same dose of P4 for 3 days. On the last day of progesterone treatment, they were given a single injection of E, (20 ng/O.l ml oil/mouse) and killed 4 and 24 h later, respectively. Mice in groups 7 and 8 were treated with cholesterol (2 mg/O.l ml oil/mouse, s.c.) or dexamethasone (200 pg/O.l ml saline/mouse, s.c.) for 2 days and killed 24 h later after the last injection. The uteri were processed as described in experiment 1 for E-2/4-H assay. Microsome preparation. Uteri were homogenized for 10 s bursts in 5 ml ice-cold TEGD buffer (10 mM Tris-HCl, 1.5 mM EDTA, 10% glycerol, and 1.5 mM DTT, pH 7.6) containing 10 ~1 5 mM E-64 and 50 ~1 10 mM phenylmethylsulphonyl fluoride in a Polytron P-10 at a dial setting of 7 (Brinkman Instrument, Westbury, NY, U.S.A.) followed by rehomogenization using a glass/Teflon homogenizer. Homogenates were centrifuged at 1000 x g for 15 min. Pellets were discarded, and supernatants were centrifuged again at 10,000 X g for 20 min to separate mitochondrial fraction. Microsomes were then prepared by centrifuging the supernatant at 105,000 X g for 1 h. Microsomes were resuspended in 5 ml TEGD buffer and recentrifuged at 105,000 x g for 1 h. The microsomal pellet was then resuspended by gentle
homogenization in 0.1 ml TEDG buffer. Microsomal protein concentrations were measured according to the method of Bradford (1976) using bovine serum albumin as a standard. Assay of E-2/4-H. Uterine microsomes were assayed for E-2/4-H by the product isolation method described previously (Chakraborty et al., 1988). In brief, the method is as follows: 50 ~1 microsome suspension (150 pg protein) was added to 100 ~1 reaction mixture containing 0.075 PCi [4-14C]Ez (10 pM), 2-OH-E, (1 PM), 4-OH-E, (1 PM), ascorbic acid (10 mM) and NADPH (1.5 mM) in Hepes/Tris (0.05 M/0.05 M) buffer (pH 7.9). The reaction was run at 30” C in a shaking water bath, and the reaction was stopped by adding 0.1 ml ice-cold 1 N HCl. Radioactive assay products were extracted twice with ethyl acetate saturated with ascorbic acid. Ethyl acetate extracts were evaporated to dryness, and the residue was reconstituted in methanol containing 1 mM ascorbic acid for HPLC analysis. Blank values were obtained by using samples not containing NADPH. The separation of CEs was performed using an LC300 liquid chromatograph equipped with a flow detector as described previously (Chakraborty et al., 1988). An IBM computer, connected with the detectors, integrated the peak areas of the separated products recorded in the 14C channel corresponding to the authentic 2/4-OH-E, in the electrochemical (EC) channel. The integrated radioactive peak areas were used for calculation of E2/4-H activity. The product was also purified and authenticated by using a neutral alumina column to adsorb the CEs and separate them from the phenolic precursors. Effects of steroidal and nonsteroidal inhibitors on E-2/4-H activity. In this study, several inhibitors were examined for their ability to interfere with uterine microsomal E-2/4-H activity. SKF525A, a-naphthoflavone, and metyrapone are cytochrome P450 inhibitors, and 2-FL-E, is a known steroidal inhibitor of E-2/4-H. The inhibitors were preincubated with the microsomal suspensions for 2 min, and the enzyme reactions run as above. Experiment 3 Effects of 2-FL-E? on implantation. In order to determine whether CEs are important for ini-
28
tiation of implantation in a Pa-primed uterus, 2a potent inhibitor (Brueggemeier and FL-E,, Kimball, 1983) and a poor substrate (Krey et al., 1983) for E-2/4-H, was used. Delayed implantation was induced by ovariectomizing pregnant mice on the morning (09.00 h) of day 4 and injecting (s.c.) daily with P4 (2 mg/O.l ml oil/mouse) beginning on day 5, for 4 days. On the final day of P4 treatment, mice were given (iv.) different doses of 2-FL-E, (25-100 ng/mouse). Animals were checked for implantation 24 h later. To determine whether effects of CEs on implantation are mediated via generation of PGs, P,-treated delayed implanting mice, prepared as above, were treated with combinations of 2-FL-E, and PGE,, and examined for implantation 24 h later. To examine the possibility that 2-FL-E, inhibits CE formation from E, and thus interferes with implantation, P,-treated delayed implanting mice were given single injections of either E, alone, or 2-FL-E, immediately followed by an injection of E,, and implantation checked 24 h later. Implantation sites were determined by an iv. injection of 0.1 ml of a 1% Chicago Blue B solution (Sigma) 5 min before the mice were killed (Psychoyos, 1973). Discrete blue bands around the uterus indicated implantation sites. Uteri of animals without implantation sites were flushed with saline to examine for the presence of blastocysts. Animals without implantation sites or blastocysts were excluded from the experiment.
NET CPM EC Scale: 30000
14C Scale: 250 0
100
100 0
25 &
t
Fig. 1. HPLC chromatograms of the catechol products produced after incubation of mouse uterine microsomal fractions with [4-r4C]E, in the presence of NADPH, followed by alumina chromatography. Left: Tracing of radiometric detection. Right: Tracing of electrochemical (EC) detection. The first peak in EC channel corresponds to that of ascorbic acid used to prevent oxidation of catechol estrogens. Peaks 1 and 2 in the radiometric channel correspond to authentic 4-OH-E, and 2-OH-E, respectively in the EC channel.
with increasing protein concentrations up to 250 pg (Fig. 3) in a reaction volume of 150 ~1. This E-4-H exhibited typical Michaelis-Menten kinetics over a range of E, concentrations from 1.5 to 11 yM (Fig. 4). Maximum velocities (V,,,) and apparent K, for the formation of 4-OH-E, were 40 pmol/mg protein/30 min and 6.8 PM, respectively. SKF-525A (325 PM), metyrapone (325 PM) and a-naphthoflavone (325 PM) inhibited uterine
1 Results Characterization of E-2/4-H Our previous studies have established that E2/4-H is maximal at pH 7.9 (Mondschein et al., 1985; Chakraborty et al., 1988). Therefore, all assays were routinely run at pH 7.9. For enzyme kinetics and inhibitor studies, assays were run in duplicate using pooled microsomes obtained from uteri of 12 mice on day 4 of pregnancy. The formation of 4-OH-E, consistently exceeded that of 2-OH-E, by about 90% (Fig. 1). Therefore, the E-2/4-H in mouse uterine microsome appears to be primarily an E-4-H. The E-4-H activity was linear for 20 min and nonlinearly increased at least up to 45 min (Fig. 2). The activity was linear
0-v
0
I
,
lo
I
,
20
I
I
30
I
,
,
40
Time (minutes)
Fig. 2. Estradiol-4-hydroxylase activity of mouse uterine microsomes as a function of time. Assays were run for 45 min using 10 PM substrate and 150 ~18 protein.
29
0
50
100
150
200
250
Protdn(~
Fig. 3. Estradiol-4-hydroxylase activity of mouse uterine microsomes as a function of protein concentration. The assays were run using 10 PM substrate for 30 mm.
Fig. 5. Estradiol-4-hydroxylase in mouse uterine microsomes during early pregnancy. Each point is the mean rt SE. Numbers in parentheses indicate the number of observations; each observation comprised of pooled uterine microsomes from two mice. Activities on different days were compared by Dunnett’s r-tests (* P i 0.01).
E-4-H activity by 72, 67 and 60%, respectively. The 2-FL-E, (50 PM) also showed a marked inhibition (82%) of E-4-H activity.
Effect of steroids As shown in not altered at 4 about 2-fold (P
Uterine E-4-H activity during the peri-implantation period The levels of E-4-H activity on days 2 through 5 are shown in Fig. 5. While the enzyme activities in the morning of days l-5 were not different from each other, the activity increased significantly (P c 0.01) in the afternoon of day 4 prior to implantation.
on uterine E-4-H activity Fig. 6, while uterine E-4-H was h (group 2) the activity increased < 0.01) when the mice were killed
TO
r” > P-
o=
(E
60
z 1
‘i
II! (3)
Y
6:: /
10 o- 1
5
Group
.’
%
0.1
0.
.
.
.
.
Fig. 4. Estradiol-4-hydroxylase activity of mouse uterine microsomes as a function of substrate concentration. Assays were run using 150 gg protein for 30 min. The insert shows the best-fit line for reciprocal of velocity vs. reciprocal of substrate concentrations. Apparent K, and V,,, for E-4-H were 6.8 PM and 40 pmol/mg protein/30 min. respectively.
.7
6
Ilk 7
(3)
6
(troatmant)
Fig. 6. Estradiol-4-hydroxylase activity in ovariectomized mouse uterine microsomes after treatment with various steroids. Group 1: no treatment; group 2: E2 (4 h): group 3: Ez (24 h); group 4: P4 (2 days); group 5: P,, (2 days) + Ez (4 h); group 6: P4 (2 days) + E, (24 h); group 7: cholesterol (2 days): group 8: dexamethasone (2 days). Numbers in parentheses indicate the number of observations: each observation comprised of pooled uterine microsomes from 2-4 mice. Statistical analyses were performed by one-way analysis of variance and Scheffe post hoc tests. Groups 3 and 4 > group 1 (* * P c 0.01) and group 6 > group 4 ( * P -c0.05).
30 TABLE
1
EFFECTS OF 2-FL-E, AND E, ON IMPLANTATION THE P,-TREATED DELAYED IMPLANTING MICE
IN
All animals received injected S.C. 2-FL-E,
P4 (2 mg/day) for 4 days. P4 and E, were was injected i.v. Values are mean f SE.
Treatment
No. of mice
No. of mice with 1,s. a
25ng 50 ng 75ng 100ng long 10 ng
6 6 9 7 5
50 ng
6
No. of IS. b
No. of blastocysts recovered ’
0 0 0 1 (14) 5 (100)
0 0 0 9 9.5kO.9
5.1 4.6 4.6 4.1 -
0
0
4.3 + 0.7
@) 2-FL-E,
E2 E2
+ 2-FL-E,
a I.S., implantation b Includes animals ’ Includes animals
TABLE
i i + *
0.5 0.7 0.5 0.5
sites. with I.S. without I.S.
2
EFFECTS OF 2-FL-E, IN THE P,-TREATED
AND PGE, ON IMPLANTATION DELAYED IMPLANTING MICE
All animals received P4 (2 mg/day) for 4 days. P4 and PGE, were injected S.C. and 2-FL-E, was injected i.v. Values are mean + SE. Treatment
No. of mice
No. of
No. of IS. a
No. of blastocysts recovered h
0
0
3.43 * 0.4
mice with IS. (%)
2-FL-E, +
10 ng
PGE, 2-FL-E, +
2/Jg 25 ng
7
PGE, 2-FL-E,
2pg 50 ng
11
3 (27)
5.8k1.6
4.0
PGE, 2-FL-E, + PGE, 2-FL-E, + PGE,
2ng 25 ng
8
8 (100)
8.5kO.9
-
5Pg 10 ng
5
5 (100)
8.0+1.1
-
5Pg
3
0
0
4.3 *0.7
a Includes h Includes
animals animals
+0.4
+
with I.S. without I.S.
24 h after a single injection of E, (group 3) or 2 days of P4 treatment (group 4) as compared to those which did not receive any steroid treatment (group 1). When 2 days of P4 treatment was combined with a single injection of E,, E-4-H activity did not increase by 4 h (group 5) but by 24 h (group 6) the activity increased 1.3-fold (P < 0.05) above that of P4 treatment only (group 4). The uterine E-4-H activity in mice treated with either cholesterol (group 7) or dexamethasone (group 8) was not different from that of the ovariectomized controls (group 1). Effect of 2-FL-E, and PGEz on embryo implantation As shown in Table 1, while a dose of 2-FL-E, as high as 100 rig/moose failed to induce implantation, a dose of E, as low as 10 rig/moose induced implantation in 100% of the animals. The implantation-inducing effect of E, (10 rig/moose)) was completely blocked by 2-FL-E, (50 ng/ mouse) when administered just prior to E, injection. Interestingly, PGE, (2-5 pg/mouse), given in combination with a dose of 2-FL-E, as low as 25 rig/moose,, induced implantation in most of the animals (Table 2). Discussion The present study provides evidence that the mouse uterus has a cytochrome P,,,-dependent monooxygenase which is capable of producing predominantly 4-OH-E, from E,. This is in contrast to most of the studies which have shown that in various tissues aromatic hydroxylation of E, at C2 position exceeds that of at C4 position (Ball and Knuppen, 1980; Bui and Weisz, 1988). Furthermore, the present observation differs from a recent report which showed that 2-hydroxylation was comparable to 4-hydroxylation in the mouse uterus (Bunyagidz and McLachlan, 1988). However, these investigators used about 30-fold more E, than the concentration of E, used in the present study for hydroxylase assay. Because 2-OH-E, and 4-OH-E, were present in our assays at the beginning of the reaction, the possibility of preferential inhibition of 2-hydroxylase over 4-hydroxylase by their products cannot be ruled out. However, in the pig blastocyst E-4-H is more sensitive
31
than E-2-H to inhibition by 2-OH-E, or 4-OH-E, (Chakraborty et al., 1988). The only other tissue which has been shown to have a monooxygenase system that exhibits predominantly E-4-H activity is the rat pituitary gland (Bui and Weisz, 1989). Our findings of the microsomal localization of the enzyme, its requirement for NADPH and inhibition by SKF-525A, metyrapone and cY-naphthoflavone provide substantial evidence that E-4-H activity in the uterus is mediated by microsomal Mitochondrial-rich fractions did P45o isozymes. not show any activity (data not shown). While SKF-525A is a reversible inhibitor of P45o (Testa and Jenner, 1981) and metyrapone is a relatively specific inhibitor of those forms of Pdso which are inducible by phenobarbitone in the liver (Testa and Jenner, 1981), a-naphthoflavone specifically inhibits cytochrome Pddgs normally inducible by aromatic hydrocarbons such as 3-methylcholanthrene (Testa and Jenner, 1981; Theron et al., 1985). Therefore, inhibition of E-4-H by cr-naphthoflavone suggests that some other form of miin the crosomal P450 may catalyze CE formation uterus. This is in contrast to the report in the pig blastocyst (Chakraborty et al., 1988) in which hydroxylation reactions are not inhibited by (Ynaphthoflavone. Previous studies have also suggested that multiple P450 isozymes are involved in CE formation (Poth et al., 1983), and direct evidence for this possibility has been found in the mouse liver (Lang and Nebert, 1981) and rat brain (Theron et al., 1985). Estrogen plays a key role in embryo implantation in the mouse and rat. Recently, we have proposed that catechol estrogens mediate some important aspects of estrogenic action in implantation (Dey and Johnson, 1986; Dey et al., 1986). Because of their unstable nature, it has been difficult to envision a role for CEs as circulating hormones (MacLusky et al., 1981; Lipsett et al., 1983). However, this would not be a consideration if they were formed locally in the blastocyst and/or uterus. It has been shown that pig blastocysts have a remarkable capacity to synthesize CEs during the time of maternal recognition of pregnancy (Mondschein et al., 1985). The present study demonstrates that the mouse uterus is capable of transforming E, into 4-OH-E,. The uterine E-4-H activity remains unaltered on the morning
of days 2, 3 and 4, but the activity showed an increase in the afternoon .of day 4 prior to implantation followed by a decline on the morning of day 5. The peak uterine activity of E-4-H on day 4 afternoon could be due to the preimplantation ovarian estrogen secretion around noon of this day (McCormack and Greenwald, 1974). Whether uterine CE formation occurs in vivo cannot be answered from this study. However, the present results suggest that CEs may be formed locally and participate in implantation. In this regard, both 2-OH-E, and 4-OH-E, are capable of inducing implantation in ovariectomized mice primed with P4 (Hoversland et al., 1982; Kantor et al., 1985) although 4-OH-E, was more potent than 2-OH-E, in this process. Other studies have also shown that estrogenic potency of 2-OH-E, is significantly lower than that of 4-OH-E, which is reported to be equipotent to the parent compound (MacLusky et al., 1981). Thus, the preferential formation of 4-OH-E, by mouse uterus may be viewed as local mediators of some of the actions of E, in implantation. The next important question is how the E-4-H activity in the uterus is regulated. Because an interaction between P4 and estrogen is essential for implantation (Psychoyos, 1973), it is conceivable that these steroids will modulate uterine E-4H activity. Indeed, an increase in E-4-H activity by P4 or E,, and an additive stimulatory effect of E, in the P,-treated uterus demonstrate that this enzyme is under the control of these steroids. The effects of P4 and E, appear to be specific, because dexamethasone or cholesterol failed to show any effect. An increased endometrial capillary permeability at the site of the blastocyst is one of the earliest prerequisite events in implantation (Psychoyos, 1973). PGs are known to participate in this process (Kennedy, 1977). CEs are more potent than phenolic estrogens in stimulating PG synthesis in both the blastocyst and endometrium in vitro (Kelly and Abel, 1981; Pakrasi and Dey, 1983). Therefore, it could be suggested that estrogen-induced implantation in a P,-primed uterus is mediated via formation of 4-OH-E, that stimulate the synthesis/release of PGs. This concept is supported by our findings of 2-FL-E, and PGE, on implantation. 2-FL-E, is a potent estrogen in
32
terms of uterotropic and other estrogenic actions (Katzenellenbogen et al., 1980; Krey et al., 1983; Liehr, 1983), but it is a poorer substrate for and a potent inhibitor of CE formation (Krey et al., 1983). This fluoroestradiol not only fails to induce implantation, but also interferes with E,-induced implantation in both the rat (Dey et al., 1986) and mouse (Table 1). However, induction of implantation by a combined treatment of 2-FL-E, and PGE, (Table 2) suggests that at least certain aspects of estrogen action in implantation are mediated via CEs and PGs. At present our knowledge regarding the mechanism of embryo implantation is limited, but the present findings suggest an autocrine/paracrine role for CEs in implantation. Acknowledgements
We thank Linda Hicks for preparation of the manuscript. This study was supported by a grant from NIH (HD12122). References Ball, P. and Knuppen, R. (1980) Acta Endocrinol. (Suppl.) 93, 1-127. Bradford, M.M. (1976) Anal. B&hem. 72, 248-254. Brueggemeier, R.W. and Kimball, J.G. (1983) Steroids 42, 93-103. Bui, Q.D. and Weisz, J. (1988) Pharmacology 36, 356-360. Bui, Q.D. and Weisz, J. (1989) Endocrinology 124, 1085-1087. Bunyagidz, C. and McLachlan, J.A. (1988) J. Steroid Biochem. 31, 795-801. Chakraborty, C., Davis, D.L. and Dey, S.K. (1988) J. Steroid B&hem. 31, 231-235. Dey, S.K. and Johnson, D.C. (1986) Ann. N.Y. Acad. Sci. 476, 49-62. Dey, SK., Johnson, D.C., Pakrasi, P.L. and Liehr, J.G. (1986) Proc. Sot. Exp. Biol. Med. 181, 215-218. Fishman, J. (1983) Annu. Rev. Physiol. 45, 61-72. Gelbke, H.P., Ball, P. and Knuppen, R. (1977) Adv. Steroid Biochem. Pharmacol. 6, 81-154.
Hoversland, R.C., Dey, S.K. and Johnson, D.C. (1982) Life Sci. 30,1801-1804. Huet, Y.M. and Dey, S.K. (1987) J. Reprod. Fertil. 81,453-458. Kantor, B.S., Dey, S.K. and Johnson, D.C. (1985) Acta Endocrinol. 109, 418-422. Katzenellenbogen, J.A., Carlson, K.E., Heiman, D.F. and Lloyd, J.E. (1980) in Structure Activity Relationship (Spencer, R.E., ed.), pp. 23-86, Grune and Stratton, New York. Kelly, R.W. and Abel, M.H. (1981) J. Steroid Biochem. 14, 787-791. Kennedy, T.G. (1977) Biol. Reprod. 16, 286-291. Krey, L.C., MacLusky, N.J., Pfeiffer, D.G., Parsons, B., Merriam, G.R. and Naftolin, F. (1983) in Catechol Estrogens (Merriam. G.R. and Lipsett, M.B., eds.), pp. 249-263, Raven Press, New York. Lang, M.A. and Nebert, D.W. (1981) J. Biol. Chem. 256, 12058-12067. Levin, M., Weisz, J., Bui. Q.D. and Santen, R. (1987) J. Steroid Biochem. 28, 513-520. Liehr, J. (1983) Mol. Pharmacol. 23, 278-281. Lipsett, M.B., Merriam, G.R., Kono, S., Brandon, D.D., Pfeiffer, D.G. and Loriaux, D.L. (1983) in Catechol Estrogens (Merriam, G.R. and Lipsett, M.B., eds.), pp. 105-114. Raven Press, New York. MacLusky, N.J., Naftolin, F., Krey, L.C. and Franks, S. (1981) J. Steroid B&hem. 15, 114-124. McCormack, J.T. and Greenwald, G.S. (1974) J. Reprod. Fertil. 41, 297-301. Mondschein, J.S., Hersey, R.M., Dey, S.K., Davis, D.L. and Weisz, J. (1985) Endocrinology 117, 2339-2346. Pakrasi, P.L. and Dey, S.K. (1983) Biol. Reprod. 29, 347-354. Paul, S.M., Axelrod, J. and Dilberto, Jr., E.J. (1977) Endocrinology 101, 1604-1610. Poth, M.A., Axelrod, J. and Hoffman, A.R. (1983) in Catechol Estrogens (Marriam, G.R. and Lipsett, M.B., eds.), pp. 19-30, Raven Press, New York. Psychoyos. A. (1973) in Handbook of Physiology (Greep, R.O., Astwood, E.G. and Geiger, S.R., eds.), pp. 187-215, American Physiological Society, Washington, DC. Schneider, J., Huh, M.M., Bradlow, H.L. and Fishman. J. (1984) J. Biol. Chem. 259, 4840-4845. Testa, B. and Jenner, P. (1981) Drug Metab. Rev. 12, l-17. Theron, C.N., Russell, V.A. and Taljaard, J.J.F. (1985) J. Steroid Biochem. 23, 919-927.
MOLCEL
25
02221
Catechol estrogen formation in the mouse uterus and its role in implantation Bibhash C. Paria, Chandan
Chakraborty
and Sudhansu
K. Dey
Department of Obstetrics and Gynecology and Physiology, University of Kansas Medical Center, Ralph L. Smith Research Center, Kansas City, KS 66103, U.S.A. (Received
Key words: Catechol
estrogen;
28 July 1989; accepted
Estradiol-2/4-hydroxylase;
31 October
Estradiol-4-hydroxylase;
1989)
Prostaglandin;
Peri-implantation;
(Mouse uterus)
Summary Estradiol-2/4-hydroxylase (E-2/4-H) activity was determined in the mouse uterus during early pregnancy as well as in ovarian steroid hormone-treated ovariectomized uterus. Under the assay conditions used, E-4-H was the predominant catechol estrogen-forming monooxygenase enzyme. The inhibition of E-4-H activity by SKF-525A, metyrapone and a-naphthoflavone suggested involvement of cytochrome P,,,-dependent monooxygenases. A haloestrogen, 2-fluoroestradiol (2-FL-E,), also inhibited this activity. During the peri-implantation period, no change in uterine E-4-H activity was noted on the morning of days 2 through 5, but the activity significantly (P < 0.01) increased in the afternoon of day 4 of pregnancy, A single injection of estradiol-17fl (E,, 100 rig/moose)) to ovariectomized mice significantly (P -=z 0.01) elevated the level of E-4-H activity at 24 h as did injections of progesterone (P4, 2 mg/mouse) for 2 days. When 2 days of P4 (2 mg/mouse) treatment was combined with a single injection of E, (20 ng/mouse), E-4-H activity increased 1.3-fold (P < 0.05) by 24 h above that of P4 treatment alone. Dexamethasone (200 pg/mouse) and cholesterol (2 mg/mouse) treatment for 2 days had no effect on E-4-H activity. Thus, the stimulatory effect of P4 and E, on E-4-H activity appeared to be specific. The increased activity of uterine E-4-H prior to implantation on day 4 evening and the modulation of its activity by Ps and/or E, suggest an involvement of 4-hydroxyestradiol in embryo implantation. In delayed implanting mice, while E, as low as 10 rig/moose induced implantation in all animals, a dose of 2-FL-E, as high as 100 rig/moose failed to induce implantation in all animals. Furthermore, administration of 2-FL-E, (50 rig/moose)) just before E, (10 rig/moose)) injection completely blocked estradiol-inducible implantation. Interestingly, administration of different doses of prostaglandin E, (PGE,, 2-5 pg/mouse) when given in combination with 2-FL-E, as low as 25 rig/moose induced implantation in most of the animals. These results suggest that at least some of the important actions of estrogen in implantation are mediated via formation of catechol estrogens and PGs.
Introduction Address for correspondence: S.K. Dey, Ph.D., Department of Obstetrics and Gynecology and Physiology, University of Kansas Medical Center, Ralph L. Smith Research Center, 39th and Rainbow Boulevard, Kansas City, KS 66103, U.S.A. 0303-7207/90/$03.50
0 1990 Elsevier Scientific
Publishers
Ireland,
In the mouse or rat, requirement for initiation Ltd.
estrogen is an absolute of embryo implantation
26
in a progesterone (P4)-primed uterus (Psychoyos, 1973; Huet and Dey, 1987). However, the mechanism by which estrogen initiates this process is not clearly understood. Recently, it has been proposed that estrogen action in implantation is mediated via local formation of catechol estrogens (CEs) in the target tissue (Hoversland et al., 1982; Kantor et al., 1985; Mondschein et al., 1985; Dey et al., 1986). CEs are the major aromatic hydroxylation products of phenolic estrogens (Fishman, 1983). The formation of CEs occurs primarily in the microsomes and is catalyzed by estradiol-2/4-hydroxylase (E-2/4-H) which is an NADPH-dependent cytochrome PdsO monooxygenase-associated enzyme (Paul et al., 1977). There are also indications that more than one form of P450s may mediate the formation of CEs (Lang and Nebert, 1981) or they could be formed by peroxidase system (Levin et al., 1987). The formation of CEs has been demonstrated to occur in a wide variety of normal and neoplastic tissues (Poth et al., 1983). CEs can bind to estrogen receptors and have biological effects as estrogen agonists or antagonists (MacLusky et al., 1981; Schneider et al., 1984). However, because of extremely short half-life and rapid clearance rate (Gelbke et al., 1977; MacLusky et al., 1981; Schneider et al., 1984) CEs are not likely to function as circulating hormones. Therefore, an attractive hypothesis is that they may act as local mediators of estrogen action in estrogen-responsive target tissues. A surge in E-2/4-H activity was noted in the pig blastocyst on days 12 and 13 of pregnancy that corresponds to the stage of pregnancy recognition (Mondschein et al., 1985). Furthermore, circumstantial evidence suggests that CEs are important for implantation in the mouse and rat (Hoversland et al., 1982; Kantor et al., 1985). However, no information is available whether the uterus and/or the embryo in these species have the capacity to form CEs during the peri-implantation period. Therefore, the temporal pattern of E-2/4-H activity was studied in the mouse uterus during this period. Since the process of implantation is primarily dependent upon an interaction between P4 and E, (Psychoyos, 1973) the effects of these steroids on uterine E-2/4-H activity were also investigated in the ovariectomized mice.
Prostaglandins (PGs) are considered to be involved in the process of implantation (Kennedy, 1977), and CEs are more potent than phenolic estrogens in stimulating PG synthesis (Kelly and Abel, 1981; Pakrasi et al., 1983). Therefore, it could be further postulated that inhibition of formation of CEs and/or PGs will interfere with implantation. Thus, effects of 2-fluoroestradiol (2FL-E,), an inhibitor of E-2/4-H, on implantation were also investigated. Materials and methods Chemicals 2-Hydroxyestradiol (2-OH-E,) and 4-hydroxyestradiol (4-OH-E,) were purchased from Steraloids (Wilton, NH, U.S.A.). [4-‘4C]E, was obtained from New England Nuclear (Boston, MA, U.S.A.). HPLC-grade methanol and sodium acetate were obtained from Fisher Scientific (St. Louis, MO, U.S.A.). Glacial acetic acid (HPLCgrade) was purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, U.S.A.). Estradiol-17/I (E2), progesterone (P4), reduced nicotinamide adenine dinucleotide phosphate (NADPH), DL-dithiothreitol (DTT), Hepes, Tris, ascorbic acid, glycerol, E-64 (trans-epoxysuccinyl-L-leucylamido-4-guanidino-butane), metyrapone (2-methyl-1,2-di-3pyridyl-propanone) and a-naphthoflavone (7,8benzoflavone) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). SKF-525A was obtained from Smith-Kline and French Laboratories (Philadelphia, PA, U.S.A.). 2-FL-E, was prepared, characterized and generously provided by Dr. J.G. Liehr (University of Texas Health Science Center, Galveston, TX). Animals CD-1 female mice (48 days old, 20-25 g, Charles River, NJ, U.S.A.) were housed in temperature(25” C) and light-controlled (14:lO h) quarters and had free access to food and water. Female mice were mated with fertile males of the same strain. The morning of finding vaginal plug was designated as day 1 of pregnancy. Experiment I To determine activity. mice
temporal pattern of uterine E-4-H were killed in the morning
27
(08.30-09.00 h) of days 1 through 5, except one group which was killed in the afternoon (15.0015.30 h) of day 4. The reproductive tracts were flushed for embryos to confirm pregnancy. The uteri were cleaned of adhering fat and mesentery, lightly blotted, weighed and kept in ice-cold TEGD buffer (see below) for E-2/4-H assay.
Experiment 2 To explore the effect of steroids on E-2/4-H activity, mice were ovariectomized regardless of their stages of the estrous cycle. After 5 days of rest, animals were divided into eight groups. Group 1 served as controls, i.e., they received only the vehicle (0.1 ml sesame seed oil). While mice in group 2 were killed 4 h after a single injection of E, (100 ng/O.l ml sesame seed oil/mouse, s.c.), those in group 3 were killed 24 h later. Mice in group 4 received injections of P4 (2 mg/O.l ml oil/mouse/day, s.c.) for 2 days and killed 24 h later. Mice in groups 5 and 6 were injected with the same dose of P4 for 3 days. On the last day of progesterone treatment, they were given a single injection of E, (20 ng/O.l ml oil/mouse) and killed 4 and 24 h later, respectively. Mice in groups 7 and 8 were treated with cholesterol (2 mg/O.l ml oil/mouse, s.c.) or dexamethasone (200 pg/O.l ml saline/mouse, s.c.) for 2 days and killed 24 h later after the last injection. The uteri were processed as described in experiment 1 for E-2/4-H assay. Microsome preparation. Uteri were homogenized for 10 s bursts in 5 ml ice-cold TEGD buffer (10 mM Tris-HCl, 1.5 mM EDTA, 10% glycerol, and 1.5 mM DTT, pH 7.6) containing 10 ~1 5 mM E-64 and 50 ~1 10 mM phenylmethylsulphonyl fluoride in a Polytron P-10 at a dial setting of 7 (Brinkman Instrument, Westbury, NY, U.S.A.) followed by rehomogenization using a glass/Teflon homogenizer. Homogenates were centrifuged at 1000 x g for 15 min. Pellets were discarded, and supernatants were centrifuged again at 10,000 X g for 20 min to separate mitochondrial fraction. Microsomes were then prepared by centrifuging the supernatant at 105,000 X g for 1 h. Microsomes were resuspended in 5 ml TEGD buffer and recentrifuged at 105,000 x g for 1 h. The microsomal pellet was then resuspended by gentle
homogenization in 0.1 ml TEDG buffer. Microsomal protein concentrations were measured according to the method of Bradford (1976) using bovine serum albumin as a standard. Assay of E-2/4-H. Uterine microsomes were assayed for E-2/4-H by the product isolation method described previously (Chakraborty et al., 1988). In brief, the method is as follows: 50 ~1 microsome suspension (150 pg protein) was added to 100 ~1 reaction mixture containing 0.075 PCi [4-14C]Ez (10 pM), 2-OH-E, (1 PM), 4-OH-E, (1 PM), ascorbic acid (10 mM) and NADPH (1.5 mM) in Hepes/Tris (0.05 M/0.05 M) buffer (pH 7.9). The reaction was run at 30” C in a shaking water bath, and the reaction was stopped by adding 0.1 ml ice-cold 1 N HCl. Radioactive assay products were extracted twice with ethyl acetate saturated with ascorbic acid. Ethyl acetate extracts were evaporated to dryness, and the residue was reconstituted in methanol containing 1 mM ascorbic acid for HPLC analysis. Blank values were obtained by using samples not containing NADPH. The separation of CEs was performed using an LC300 liquid chromatograph equipped with a flow detector as described previously (Chakraborty et al., 1988). An IBM computer, connected with the detectors, integrated the peak areas of the separated products recorded in the 14C channel corresponding to the authentic 2/4-OH-E, in the electrochemical (EC) channel. The integrated radioactive peak areas were used for calculation of E2/4-H activity. The product was also purified and authenticated by using a neutral alumina column to adsorb the CEs and separate them from the phenolic precursors. Effects of steroidal and nonsteroidal inhibitors on E-2/4-H activity. In this study, several inhibitors were examined for their ability to interfere with uterine microsomal E-2/4-H activity. SKF525A, a-naphthoflavone, and metyrapone are cytochrome P450 inhibitors, and 2-FL-E, is a known steroidal inhibitor of E-2/4-H. The inhibitors were preincubated with the microsomal suspensions for 2 min, and the enzyme reactions run as above. Experiment 3 Effects of 2-FL-E? on implantation. In order to determine whether CEs are important for ini-
28
tiation of implantation in a Pa-primed uterus, 2a potent inhibitor (Brueggemeier and FL-E,, Kimball, 1983) and a poor substrate (Krey et al., 1983) for E-2/4-H, was used. Delayed implantation was induced by ovariectomizing pregnant mice on the morning (09.00 h) of day 4 and injecting (s.c.) daily with P4 (2 mg/O.l ml oil/mouse) beginning on day 5, for 4 days. On the final day of P4 treatment, mice were given (iv.) different doses of 2-FL-E, (25-100 ng/mouse). Animals were checked for implantation 24 h later. To determine whether effects of CEs on implantation are mediated via generation of PGs, P,-treated delayed implanting mice, prepared as above, were treated with combinations of 2-FL-E, and PGE,, and examined for implantation 24 h later. To examine the possibility that 2-FL-E, inhibits CE formation from E, and thus interferes with implantation, P,-treated delayed implanting mice were given single injections of either E, alone, or 2-FL-E, immediately followed by an injection of E,, and implantation checked 24 h later. Implantation sites were determined by an iv. injection of 0.1 ml of a 1% Chicago Blue B solution (Sigma) 5 min before the mice were killed (Psychoyos, 1973). Discrete blue bands around the uterus indicated implantation sites. Uteri of animals without implantation sites were flushed with saline to examine for the presence of blastocysts. Animals without implantation sites or blastocysts were excluded from the experiment.
NET CPM EC Scale: 30000
14C Scale: 250 0
100
100 0
25 &
t
Fig. 1. HPLC chromatograms of the catechol products produced after incubation of mouse uterine microsomal fractions with [4-r4C]E, in the presence of NADPH, followed by alumina chromatography. Left: Tracing of radiometric detection. Right: Tracing of electrochemical (EC) detection. The first peak in EC channel corresponds to that of ascorbic acid used to prevent oxidation of catechol estrogens. Peaks 1 and 2 in the radiometric channel correspond to authentic 4-OH-E, and 2-OH-E, respectively in the EC channel.
with increasing protein concentrations up to 250 pg (Fig. 3) in a reaction volume of 150 ~1. This E-4-H exhibited typical Michaelis-Menten kinetics over a range of E, concentrations from 1.5 to 11 yM (Fig. 4). Maximum velocities (V,,,) and apparent K, for the formation of 4-OH-E, were 40 pmol/mg protein/30 min and 6.8 PM, respectively. SKF-525A (325 PM), metyrapone (325 PM) and a-naphthoflavone (325 PM) inhibited uterine
1 Results Characterization of E-2/4-H Our previous studies have established that E2/4-H is maximal at pH 7.9 (Mondschein et al., 1985; Chakraborty et al., 1988). Therefore, all assays were routinely run at pH 7.9. For enzyme kinetics and inhibitor studies, assays were run in duplicate using pooled microsomes obtained from uteri of 12 mice on day 4 of pregnancy. The formation of 4-OH-E, consistently exceeded that of 2-OH-E, by about 90% (Fig. 1). Therefore, the E-2/4-H in mouse uterine microsome appears to be primarily an E-4-H. The E-4-H activity was linear for 20 min and nonlinearly increased at least up to 45 min (Fig. 2). The activity was linear
0-v
0
I
,
lo
I
,
20
I
I
30
I
,
,
40
Time (minutes)
Fig. 2. Estradiol-4-hydroxylase activity of mouse uterine microsomes as a function of time. Assays were run for 45 min using 10 PM substrate and 150 ~18 protein.
29
0
50
100
150
200
250
Protdn(~
Fig. 3. Estradiol-4-hydroxylase activity of mouse uterine microsomes as a function of protein concentration. The assays were run using 10 PM substrate for 30 mm.
Fig. 5. Estradiol-4-hydroxylase in mouse uterine microsomes during early pregnancy. Each point is the mean rt SE. Numbers in parentheses indicate the number of observations; each observation comprised of pooled uterine microsomes from two mice. Activities on different days were compared by Dunnett’s r-tests (* P i 0.01).
E-4-H activity by 72, 67 and 60%, respectively. The 2-FL-E, (50 PM) also showed a marked inhibition (82%) of E-4-H activity.
Effect of steroids As shown in not altered at 4 about 2-fold (P
Uterine E-4-H activity during the peri-implantation period The levels of E-4-H activity on days 2 through 5 are shown in Fig. 5. While the enzyme activities in the morning of days l-5 were not different from each other, the activity increased significantly (P c 0.01) in the afternoon of day 4 prior to implantation.
on uterine E-4-H activity Fig. 6, while uterine E-4-H was h (group 2) the activity increased < 0.01) when the mice were killed
TO
r” > P-
o=
(E
60
z 1
‘i
II! (3)
Y
6:: /
10 o- 1
5
Group
.’
%
0.1
0.
.
.
.
.
Fig. 4. Estradiol-4-hydroxylase activity of mouse uterine microsomes as a function of substrate concentration. Assays were run using 150 gg protein for 30 min. The insert shows the best-fit line for reciprocal of velocity vs. reciprocal of substrate concentrations. Apparent K, and V,,, for E-4-H were 6.8 PM and 40 pmol/mg protein/30 min. respectively.
.7
6
Ilk 7
(3)
6
(troatmant)
Fig. 6. Estradiol-4-hydroxylase activity in ovariectomized mouse uterine microsomes after treatment with various steroids. Group 1: no treatment; group 2: E2 (4 h): group 3: Ez (24 h); group 4: P4 (2 days); group 5: P,, (2 days) + Ez (4 h); group 6: P4 (2 days) + E, (24 h); group 7: cholesterol (2 days): group 8: dexamethasone (2 days). Numbers in parentheses indicate the number of observations: each observation comprised of pooled uterine microsomes from 2-4 mice. Statistical analyses were performed by one-way analysis of variance and Scheffe post hoc tests. Groups 3 and 4 > group 1 (* * P c 0.01) and group 6 > group 4 ( * P -c0.05).
30 TABLE
1
EFFECTS OF 2-FL-E, AND E, ON IMPLANTATION THE P,-TREATED DELAYED IMPLANTING MICE
IN
All animals received injected S.C. 2-FL-E,
P4 (2 mg/day) for 4 days. P4 and E, were was injected i.v. Values are mean f SE.
Treatment
No. of mice
No. of mice with 1,s. a
25ng 50 ng 75ng 100ng long 10 ng
6 6 9 7 5
50 ng
6
No. of IS. b
No. of blastocysts recovered ’
0 0 0 1 (14) 5 (100)
0 0 0 9 9.5kO.9
5.1 4.6 4.6 4.1 -
0
0
4.3 + 0.7
@) 2-FL-E,
E2 E2
+ 2-FL-E,
a I.S., implantation b Includes animals ’ Includes animals
TABLE
i i + *
0.5 0.7 0.5 0.5
sites. with I.S. without I.S.
2
EFFECTS OF 2-FL-E, IN THE P,-TREATED
AND PGE, ON IMPLANTATION DELAYED IMPLANTING MICE
All animals received P4 (2 mg/day) for 4 days. P4 and PGE, were injected S.C. and 2-FL-E, was injected i.v. Values are mean + SE. Treatment
No. of mice
No. of
No. of IS. a
No. of blastocysts recovered h
0
0
3.43 * 0.4
mice with IS. (%)
2-FL-E, +
10 ng
PGE, 2-FL-E, +
2/Jg 25 ng
7
PGE, 2-FL-E,
2pg 50 ng
11
3 (27)
5.8k1.6
4.0
PGE, 2-FL-E, + PGE, 2-FL-E, + PGE,
2ng 25 ng
8
8 (100)
8.5kO.9
-
5Pg 10 ng
5
5 (100)
8.0+1.1
-
5Pg
3
0
0
4.3 *0.7
a Includes h Includes
animals animals
+0.4
+
with I.S. without I.S.
24 h after a single injection of E, (group 3) or 2 days of P4 treatment (group 4) as compared to those which did not receive any steroid treatment (group 1). When 2 days of P4 treatment was combined with a single injection of E,, E-4-H activity did not increase by 4 h (group 5) but by 24 h (group 6) the activity increased 1.3-fold (P < 0.05) above that of P4 treatment only (group 4). The uterine E-4-H activity in mice treated with either cholesterol (group 7) or dexamethasone (group 8) was not different from that of the ovariectomized controls (group 1). Effect of 2-FL-E, and PGEz on embryo implantation As shown in Table 1, while a dose of 2-FL-E, as high as 100 rig/moose failed to induce implantation, a dose of E, as low as 10 rig/moose induced implantation in 100% of the animals. The implantation-inducing effect of E, (10 rig/moose)) was completely blocked by 2-FL-E, (50 ng/ mouse) when administered just prior to E, injection. Interestingly, PGE, (2-5 pg/mouse), given in combination with a dose of 2-FL-E, as low as 25 rig/moose,, induced implantation in most of the animals (Table 2). Discussion The present study provides evidence that the mouse uterus has a cytochrome P,,,-dependent monooxygenase which is capable of producing predominantly 4-OH-E, from E,. This is in contrast to most of the studies which have shown that in various tissues aromatic hydroxylation of E, at C2 position exceeds that of at C4 position (Ball and Knuppen, 1980; Bui and Weisz, 1988). Furthermore, the present observation differs from a recent report which showed that 2-hydroxylation was comparable to 4-hydroxylation in the mouse uterus (Bunyagidz and McLachlan, 1988). However, these investigators used about 30-fold more E, than the concentration of E, used in the present study for hydroxylase assay. Because 2-OH-E, and 4-OH-E, were present in our assays at the beginning of the reaction, the possibility of preferential inhibition of 2-hydroxylase over 4-hydroxylase by their products cannot be ruled out. However, in the pig blastocyst E-4-H is more sensitive
31
than E-2-H to inhibition by 2-OH-E, or 4-OH-E, (Chakraborty et al., 1988). The only other tissue which has been shown to have a monooxygenase system that exhibits predominantly E-4-H activity is the rat pituitary gland (Bui and Weisz, 1989). Our findings of the microsomal localization of the enzyme, its requirement for NADPH and inhibition by SKF-525A, metyrapone and cY-naphthoflavone provide substantial evidence that E-4-H activity in the uterus is mediated by microsomal Mitochondrial-rich fractions did P45o isozymes. not show any activity (data not shown). While SKF-525A is a reversible inhibitor of P45o (Testa and Jenner, 1981) and metyrapone is a relatively specific inhibitor of those forms of Pdso which are inducible by phenobarbitone in the liver (Testa and Jenner, 1981), a-naphthoflavone specifically inhibits cytochrome Pddgs normally inducible by aromatic hydrocarbons such as 3-methylcholanthrene (Testa and Jenner, 1981; Theron et al., 1985). Therefore, inhibition of E-4-H by cr-naphthoflavone suggests that some other form of miin the crosomal P450 may catalyze CE formation uterus. This is in contrast to the report in the pig blastocyst (Chakraborty et al., 1988) in which hydroxylation reactions are not inhibited by (Ynaphthoflavone. Previous studies have also suggested that multiple P450 isozymes are involved in CE formation (Poth et al., 1983), and direct evidence for this possibility has been found in the mouse liver (Lang and Nebert, 1981) and rat brain (Theron et al., 1985). Estrogen plays a key role in embryo implantation in the mouse and rat. Recently, we have proposed that catechol estrogens mediate some important aspects of estrogenic action in implantation (Dey and Johnson, 1986; Dey et al., 1986). Because of their unstable nature, it has been difficult to envision a role for CEs as circulating hormones (MacLusky et al., 1981; Lipsett et al., 1983). However, this would not be a consideration if they were formed locally in the blastocyst and/or uterus. It has been shown that pig blastocysts have a remarkable capacity to synthesize CEs during the time of maternal recognition of pregnancy (Mondschein et al., 1985). The present study demonstrates that the mouse uterus is capable of transforming E, into 4-OH-E,. The uterine E-4-H activity remains unaltered on the morning
of days 2, 3 and 4, but the activity showed an increase in the afternoon .of day 4 prior to implantation followed by a decline on the morning of day 5. The peak uterine activity of E-4-H on day 4 afternoon could be due to the preimplantation ovarian estrogen secretion around noon of this day (McCormack and Greenwald, 1974). Whether uterine CE formation occurs in vivo cannot be answered from this study. However, the present results suggest that CEs may be formed locally and participate in implantation. In this regard, both 2-OH-E, and 4-OH-E, are capable of inducing implantation in ovariectomized mice primed with P4 (Hoversland et al., 1982; Kantor et al., 1985) although 4-OH-E, was more potent than 2-OH-E, in this process. Other studies have also shown that estrogenic potency of 2-OH-E, is significantly lower than that of 4-OH-E, which is reported to be equipotent to the parent compound (MacLusky et al., 1981). Thus, the preferential formation of 4-OH-E, by mouse uterus may be viewed as local mediators of some of the actions of E, in implantation. The next important question is how the E-4-H activity in the uterus is regulated. Because an interaction between P4 and estrogen is essential for implantation (Psychoyos, 1973), it is conceivable that these steroids will modulate uterine E-4H activity. Indeed, an increase in E-4-H activity by P4 or E,, and an additive stimulatory effect of E, in the P,-treated uterus demonstrate that this enzyme is under the control of these steroids. The effects of P4 and E, appear to be specific, because dexamethasone or cholesterol failed to show any effect. An increased endometrial capillary permeability at the site of the blastocyst is one of the earliest prerequisite events in implantation (Psychoyos, 1973). PGs are known to participate in this process (Kennedy, 1977). CEs are more potent than phenolic estrogens in stimulating PG synthesis in both the blastocyst and endometrium in vitro (Kelly and Abel, 1981; Pakrasi and Dey, 1983). Therefore, it could be suggested that estrogen-induced implantation in a P,-primed uterus is mediated via formation of 4-OH-E, that stimulate the synthesis/release of PGs. This concept is supported by our findings of 2-FL-E, and PGE, on implantation. 2-FL-E, is a potent estrogen in
32
terms of uterotropic and other estrogenic actions (Katzenellenbogen et al., 1980; Krey et al., 1983; Liehr, 1983), but it is a poorer substrate for and a potent inhibitor of CE formation (Krey et al., 1983). This fluoroestradiol not only fails to induce implantation, but also interferes with E,-induced implantation in both the rat (Dey et al., 1986) and mouse (Table 1). However, induction of implantation by a combined treatment of 2-FL-E, and PGE, (Table 2) suggests that at least certain aspects of estrogen action in implantation are mediated via CEs and PGs. At present our knowledge regarding the mechanism of embryo implantation is limited, but the present findings suggest an autocrine/paracrine role for CEs in implantation. Acknowledgements
We thank Linda Hicks for preparation of the manuscript. This study was supported by a grant from NIH (HD12122). References Ball, P. and Knuppen, R. (1980) Acta Endocrinol. (Suppl.) 93, 1-127. Bradford, M.M. (1976) Anal. B&hem. 72, 248-254. Brueggemeier, R.W. and Kimball, J.G. (1983) Steroids 42, 93-103. Bui, Q.D. and Weisz, J. (1988) Pharmacology 36, 356-360. Bui, Q.D. and Weisz, J. (1989) Endocrinology 124, 1085-1087. Bunyagidz, C. and McLachlan, J.A. (1988) J. Steroid Biochem. 31, 795-801. Chakraborty, C., Davis, D.L. and Dey, S.K. (1988) J. Steroid B&hem. 31, 231-235. Dey, S.K. and Johnson, D.C. (1986) Ann. N.Y. Acad. Sci. 476, 49-62. Dey, SK., Johnson, D.C., Pakrasi, P.L. and Liehr, J.G. (1986) Proc. Sot. Exp. Biol. Med. 181, 215-218. Fishman, J. (1983) Annu. Rev. Physiol. 45, 61-72. Gelbke, H.P., Ball, P. and Knuppen, R. (1977) Adv. Steroid Biochem. Pharmacol. 6, 81-154.
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