Life Sciences, Vol. 53, pp. 1625-1630
`Printed in the USA
`
`Pergamon Press
`
`CONTRASTING ACTION OF ANTIESTROGEN (ICI—182780) FOR PREVENTING INITIATION
`OF EMBRYO IMPLANTATION BY ESTRADIOL OR EPIDERMAL GROWTH FACTOR (EGF)
`
`S.Chatterjee and D.C.Johnson
`
`Depts of Gynecology & Obstetrics and Physiology
`R.L.Smith Research Center
`University of Kansas Medical Center, Kansas City, KS 66160
`
`(Received in final form September 15, 1993)
`
`Summary
`
`The pure estrogen antagonist ICI-182780, at doses above 50 [Lg/kg,
`effectively inhibited the initiation of embryo implantation in rats
`when administered on day 4 of pregnancy (day l=sperm positive). The
`same dose inhibited the implantation initiating effect of intrave-
`nous 25 ng of estradiol—17S in delayed implanting progesterone-
`primed hypophysectomized rats.
`In contrast,
`the anti—estrogen at a
`dose of
`1 mg/kg was
`ineffective at
`inhibiting the initiation of
`implantation induced by intrauterine plus intravenous administration
`of murine epidermal growth factor to delayed implanting rats. The
`growth factor also initiated implantation of blastocysts transferred
`from donor animals injected with the anti-estrogen to progesterone—
`primed hypophysectomized recipients. The results clearly demonstrate
`that the implantation initiating effect of the growth factor is not
`inhibited by
`a pure estrogen antagonist,
`and therefore this
`estrogenic function does not appear to require action initiated by
`the classical estrogen receptor.
`
`Although not clearly defined, an action of estrogen on the progesterone—
`primed uterus is necessary for initiating implantation of the embryo into the
`rodent uterus
`(reviewed in l
`& 2). Recent evidence indicates that estrogen
`action, at least as applied to mitogenic activity of the steroid, may involve
`synthesis of growth factors and their receptors, particularly epidermal growth
`factor (EGF) and transforming growth factor alpha (TGFa)(3). Furthermore there
`is one report
`that exogenous administration of EGF mimicked the effect of
`estrogen on the uterine and vaginal epithelium of the mouse (4). This effect of
`EGF was attenuated by co-administration of
`a
`so—called “pure",
`i.e. only
`antagonistic action, anti—estrogen (ICI-l64,384)(5). other studies have also
`shown that anti—estrogens can reduce or inhibit the effect of growth factors,
`suggesting an
`interaction between the signal
`transduction system of
`the
`classical estrogen receptor and the growth factors (5,6).
`
`Recently we have shown that exogenous EGF can replace estrogen for the
`initiation of embryo implantation in the rat (7,8). We do not know whether this
`implantation—initiating effect of EGF is related to its mitogenic action, but
`the apparent
`requirement
`for arachidonic acid metabolites for both effects
`(8,9,10)
`suggests
`a
`similarity.
`Increases
`in arachidonic
`acid and
`its
`metabolites has long been associated with the estrogenic action involved with
`embryo implantation (1,2). With these relationships in mind we
`tested the
`ability of a 'pure' anti—estrogen (ICI—182,780), which is structurally similar
`to that of ICI—164,384 (11),
`to inhibit the initiation of implantation brought
`about by EGF in the delayed implanting, progesterone—primed, hypophysectomized
`rat model. The results clearly demonstrate that while the anti—estrogen inhibits
`the implantation associated with endogenous or exogenous estradiol it does not
`inhibit this action of EGF.
`
`Methods
`
`Chemicals: Progesterone, estradiol—17B, culture medium and bovine serum albumin
`were obtained from Sigma Chemical Co (St. Louis, M0.). Receptor grade murine
`epidermal growth factor (EGF) was purchased from Harlan Bioproducts for Science
`
`0024-3205/93 $6.00 + .00
`Copyright 0 1993 Pergamon Press Ltd All rights reserved.
`
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`

`1626
`
`Estrogen, EGF and Embryo Implantation
`
`Vol. 53, No. 21, 1993
`
`IN) and was dissolved in sterile phosphate—buffered (pH 7.4)
`(Indianapolis,
`saline just prior
`to use. The anti—estrogen ICI-182,780 [7a(9-(4,4,5,5,5—
`pentafluoropentylsulfinyl)nonyl)estra—l,3,S(lO)-triene—3,l7B—diol] was provided
`by ICI Pharmaceuticals (Cheshire, UK),
`through the courtesy of Dr. B. Vose, and
`was dissolved in sesame seed oil: benzyl benzoate 80:20 (v/v).
`
`the Holtzman strain
`rats of
`(225i25 9)
`Animals: Young adult virgin female
`(Harlan, Sprague—Dawley, Madison, WI) were maintained in temperature (2311°C)
`and light (lights on 0600—2000 h) controlled quarters with free access to Purina
`Laboratory Chow and tap water. On the afternoon of proestrus, as determined by
`examination of the vaginal
`lavage, a female was placed in the home cage of two
`proven male breeders of the same strain. Presence of vaginal spermatozoa on the
`following morning was
`taken as day 1 of pregnancy. Delayed implantation was
`induced by hypophysectomy, using the parapharyngeal approach with ether anesthe—
`sia, on the morning of day 3. Following this operation the animals were provided
`with 5% glucose water and softened food. Pregnancy was maintained in the
`hypophysectomized animals by daily injection (so) of 2 mg progesterone dissolved
`in 0.1ml of
`sesame seed oilzbenzyl benzoate (80:20, v/v). Females used as
`recipients for embryo transfers were hypophysectomized without regard to the
`stage of
`the estrous cycle. These animals were untreated for 72
`h before
`receiving (sc)
`2 mg progesterone daily for at
`least 2 days before receiving
`embryos. On the day of embryo transfer the hair was
`removed from the flank
`region on one side,
`the skin washed with alcohol and an incision made through
`the skin and peritoneum. After exposure of a uterine horn blastocysts were
`transferred with a micropipette into the lumen via a puncture made with a
`sterile 25 gauge needle. After replacing the uterus within the peritoneum the
`skin was closed with 11 mm wound clips. The blastocysts for
`transfer were
`obtained by flushing the uteri of normal day 5 pregnant rats with Dulbecco’s
`modified Eagle's medium containing 1% bovine serum albumin (RIA grade) but no
`phenol red. The blastocysts were washed in the same medium before transfer.
`
`For initiating implantation with EGF animals were anesthetized with ether
`and one uterine horn exposed as described above. Murine EGF (1.5ug in Bul) was
`injected into the uterine lumen using a lOul syringe with a fixed needle (Hamil—
`ton Company Reno NV),
`the uterus returned in the peritoneum and the wound closed
`with 11 mm clips. Two h later 0.2ml of saline containing 100 ug of the same EGF
`was injected via a lateral tail vein using light ether anesthesia. Forty-eight
`h later the animals were anesthetized with ether and injected (iv) with 0.5ml
`of a 1% solution of Chicago Blue B in 0.15 molar NaCl 15 min before killing with
`an overdose of ether. For testing the effect of ICI—182 on normal implantation
`the animals were injected (so) with the anti—estrogen on the morning of day 4
`and injected with the macromolecular dye on the morning of day 6. The uteri were
`examined for implantation sites, which were evident at places of embryo—uterine
`interaction due to increased capillary permeability (1).
`If no sites were
`present the uterine horns were flushed with saline to determine the presence and
`number of blastocysts. If neither sites nor blastocysts were found the animal
`was assumed not to be pregnant and was discarded from the study.
`
`reviewed and approved by the
`involving animals were
`All procedures
`University of Kansas Medical Center Animal Care and Use Committee and followed
`guidelines set
`forth by the Public Health Service for the care and use of
`laboratory animals.
`
`m
`
`The effect of giving ICI—182 on day 4 of pregnancy on implantation is
`shown in Table I. The anti—estrogen inhibits the effects of endogenous estrogen
`at a dose of about Song/kg. Even with a dose as large as 1 mg/kg blastocysts
`were recovered in the uteri 7 days later;
`implantation could not be initiated
`in these animals by 100 ng of estradiol benzoate given (so) 48 h earlier.
`
`Delayed implanting hypophysectomized control animals (group 1, Table II)
`received an intrauterine injection of 3u1 of saline followed 2 h later by an iv
`injection of 0.2 ml saline. Although all animals had blastocysts there were no
`implantation sites. Thus the trauma of anesthesia, surgery, and intrauterine
`injection was
`insufficient
`to initiate implantation in progesterone—primed
`animals. In contrast,
`the animals of group 2, treated with a single iv injection
`of 25 ng of estradiol in 0.25 ml saline, had 50 implantation sites. This effect
`of estradiol, however, could be prevented by subcutaneous injection of 0.1mg/kg
`ICI—182 one h before giving the estrogen (group 3). The addition of intrauterine
`
`AstraZeneca Exhibit 2161 p. 2
`
`

`

`Vol. 53, No. 21, 1993
`
`Estrogen, EGF and Embryo Implantation
`
`1627
`
`trauma by injecting 3111 of saline and the injection of 0.2m1 of saline 2 h later
`did not alter the inhibiting action of
`ICI—182 upon estradiol
`(group 3A).
`Implantation sites were missing also in animals that received 0.05 mg/kg ICI—182
`one h before estradiol
`(group 4). Reducing the dose of
`ICI
`to 0.025mg/kg
`resulted in implantation being initiated in 2 of
`the 5 rats treated with
`estradiol
`(group 5) .
`
`TABLE I
`
`Inhibition of Implantation by the Anti-estrogen ICI-182,780.
`Dose of ICI—182
`Number with sites/
`Number of
`(mg/kg bd wt)
`Total number
`Blastocysts or Implant sites/rat
`
`0
`10.81l.0
`0/6
`0.5
`0
`14
`0/2
`0.25
`0
`11
`0/2
`0.125
`0
`10.5
`0/2
`0.0625
`0
`11
`0/2
`0.047
`15
`0
`2/2
`0.03125
`12
`0
`2/2
`0.0156
`(sperm+ = day l) and
`Pregnant rats were injected (sc) on the morning of day 2
`given 0.5m1 of a 1% solution of Chicago Blue B 15 min before autopsy on the
`morning of day 6.
`* Blastocysts were recovered on day 11 and had not
`implanted
`after the animals received 100 ng of estradiol benzoate in oil
`(sc) on day 9.
`
`Intrauterine and intravenous dosing with EGF initiated 31 implantation
`sites in the 9 rats of group 6 (Table II). Administration of 1 mg/kg ICI—182 one
`h before the intrauterine and 3 h before the intravenous injection of EGF
`resulted in 50 sites in 8 of the 10 animals of group 7. The same doses of EGF
`initiated 22 sites in 4 of 5 rats in which the effect of 25ng of estradiol had
`been inhibited by 0.1 mg/kg ICI—182 (compare groups 3 and 8).
`
`TABLE II
`
`Effect of Antiestrogen on Initiation of Implantation Induced by Epidermal Growth
`Factor (EGF) .
`ICI-182 EGF
`Group
`No of
`animals mg/kg
`pg
`
`E2
`ng
`
`0.1
`100
`Rats were hypophysectomized on day 3 of pregnancy (day 1: sperm positive) and
`given 2mg progesterone (sc) daily. After 6 days of delay ICI~182,780 dissolved
`in oil was
`injected (sc)
`1 h before estradiol
`(E2) 25ng (iv)
`in saline or
`intrauterine injection of 1.5},Lg (3111) EGF and 3 h before 100p.g (0.2 ml,) EGF in
`saline (iv). Animals in groups 1 and 3A (#) received intrauterine and iv saline.
`Implantation sites were counted 48 h after treatment and visualized 15 min after
`injection of 0.5ml of Chicago Blue B in normal saline. Animals without implanta—
`tion sites or blastocysts were discarded as not pregnant.
`
`1
`
`3
`3A
`4
`
`6
`
`7
`
`5
`5
`3
`3
`4
`5
`9
`
`0
`0
`0.
`0.1
`0.05
`0.025
`0
`
`10
`
`1
`
`8
`
`5
`
`0
`
`25
`25#
`25
`25
`0
`
`0
`
`0
`0
`0
`0
`0
`0
`1.5+
`100
`1.5+
`100
`1.5+ 25
`
`No with implant No of Blastocysts
`sites/total No
`sites
`(percent)
`0
`(0)
`5 (100)
`0
`(0)
`0
`(0)
`0
`(0)
`2 (40)
`9 (100)
`
`.__ .__——
`7.211.1
`———
`9.310.7
`9.711.4
`8.211.4
`7.71l.2
`———
`
`0
`50
`0
`0
`0
`9
`31
`
`8 (80)
`
`50
`
`15,2
`
`4
`
`(80)
`
`22 6
`
`In preliminary experiments blastocysts from normal day 5 pregnant rats
`were transferred to the uteri of progesterone—primed hypophysectomized animals
`
`AstraZeneca Exhibit 2161 p. 3
`
`

`

`1628
`
`Estrogen, EGF and Embryo Implantation
`
`Vol. 53, No. 21, 1993
`
`that had received 1mg/kg ICI—182 one h before transfer. Only 2 of 9 animals had
`a total of 7 sites but 33 of 74 (44%) of the transferred embryos were recovered.
`In those studies, however,
`the iv dose of EGF was given at
`the time of the
`embryo transfer.
`Subsequent experiments revealed that EGF was more effective
`when given after a period of
`recovery from an intra—uterine injection and
`therefore the study was repeated using such a regimen.
`TABLE III
`
`Implantation of Embryos Transferred to Progesterone-primed
`Initiation of
`Hypophysectomized Recipients Treated with EGF.
`Group
`Treatment of
`Number with sites/ Number of sites/total
`Recipient
`Donor
`total number
`number transferred
`ECF
`Oil
`Z75 (EU?)
`11742l26.2¥)
`
`1
`
`2
`3
`
`EGF
`ICI lmg/kg+
`EGF
`
`ICI 1mg/kg
`———
`
`4/5 (80%)
`4/5 (80%)
`
`18/39(46.1%)
`l9/56(33.9%)
`
`Realpienfs received progesEerone (2mgFanJ for aE least 2 days Before Eransfer.
`ICI-182, dissolved in oil, was given to the recipient one h before embryo
`transfer, but it was given to the donor on the morning of day 4 of pregnancy.
`Blastocysts were harvested from donor animals on the morning of day 5 of
`pregnancy. EGF was injected (iv) 2 h after embryo transfer.
`
`the 5 hypophysectomized progesterone—primed recipients that
`Four of
`received day 5 blastocysts from donors that were injected (so) with oil one h
`before transfer had implantation sites (group 1, table III). The same result was
`obtained when the blastocysts were recovered from donor animals that had been
`treated with ICI-182 (1 mg/kg) 24 h earlier (group 2). The percentage of sites
`per transfer was somewhat
`improved in this group. Injection of ICI—182 to the
`recipient one h before it received day 5 blastocysts from normal untreated
`donors did not prevent initiation of implantation in 4 of the 5 animals injected
`with EGF.
`
`W
`
`The present results clearly demonstrate that ICI—182 effectively blocks
`initiation of implantation when administered in a dose of about SOug/kg on day
`4 of pregnancy, before the time of
`the presumed endogenous estrogen surge
`(discussed in 2). On the other hand 1 mg/kg of the anti—estrogen given on the
`morning of day 5 or day 6 had no effect upon implantation (data not shown).
`In
`the delayed implanting hypophysectomized rat model, which is extremely sensitive
`to exogenous estrogen (12), about SOug/kg ICI-182 blocked the effect of 25ng
`(=100ng/kg) estradiol even when given only one h in advance of the latter.
`Although some controversy exits concerning the mechanism of action of ICI—182
`(13)
`there is universal agreement that it functions by competitive binding to
`the nuclear estrogen receptor
`and inhibits the activation by the estrogen
`response element.
`
`Intravenous administration of EGF initiates implantation in the delayed
`implanting rat model if the uterus is also exposed to a small amount of trauma
`(7,8). The latter can be provided either by the technique of embryo transfer or
`by intrauterine injection. The uterine trauma can be omitted if multiple doses
`of prostaglandins (PGE2 or PGF2a) or histamine dihydrochloride are administered
`(ip) before and after EGF (Chatterjee and Johnson, unpublished data). On the
`other hand,
`injection of the cyclooxygenase inhibitor indomethacin (8) but not
`the lipoxygenase inhibitor nordihydroguaiaretic acid (Chatterjee and Johnson,
`unpublished data)
`inhibits the effect of EGF on implantation suggesting that
`prostaglandins are required for the response. The mitogenic activity of EGF in
`BALB/c3T3 cells also requires
`the production of prostaglandin (9),
`but
`leukotrienes appear to be involved with EGF action in A431 cells (10).
`
`The actions of several growth factors have been shown to be inhibited by
`anti-estrogens.
`Increases in progesterone receptor, an estrogenic response in
`the rat uterus that is also induced by cyclic AMP and insulin-like growth factor
`I was inhibited by ICI—164384 (6). The mitogenic activity of EGF in reproductive
`tract tissues was inhibited by the same anti—estrogen (5). However, conflicting
`reports regarding the effects of anti—estrogens on growth factor action in
`breast cancer cells have appeared. Vignon et a1
`(14)
`reported that anti—
`
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`
`

`

`Vol. 53, No. 21, 1993
`
`Estrogen, EGF and Embryo Implantation
`
`1629
`
`estrogens inhibited the mitogenic action of EGF and IGF—1 on MCF—7 breast cancer
`cells. These results were recently confirmed (15).
`In contrast, Cormier and
`Jordan (16) reported that anti—estrogens,
`tamoxifen or ICI—164384, had no effect
`upon the mitogenic action of EGF in MCF—7 cells. The latter authors also found
`that with mixtures of EGF and estradiol, only the action of the steroid was
`inhibited by the anti—estrogens.
`
`ICI—182 certainly did not inhibit the implantation initiating effect of
`EGF in the present experiments. Actually there is a suggestion that the anti-
`estrogen enhanced the action of EGF. Comparing the results of group 6 and 7
`(Table II) it is apparent that with EGF alone the average number of implantation
`sites per rat was 3.4 but when
`ICI—182 was administered an hour before
`intrauterine EGF the rate was 6.2. Furthermore, only 1 of the 9 rats that did
`not receive ICI-182 had implantation sites in both uterine horns,
`i.e.
`the
`injected and non—injected sides, whereas with ICI-182 5 of the 8 animals had
`implantations in both horns.
`
`shown that estrogenic effects on the uterine
`studies have
`Previous
`epithelium and the embryo trophoblast are required for initiating implantation
`(12). That is, delayed implanting embryos transferred into a progesterone—primed
`uterus that had been exposed to intravenous estradiol one h earlier do not
`implant. If on the other hand,
`the embryos were obtained from a uterus that had
`been exposed to estrogen one h before transfer
`then implantation occurs.
`Intravenously administered EGF is also more effective at initiating implantation
`of transferred blastocysts obtained one h after the donor received estradiol
`(7).
`In the present study non-delayed embryos,
`i.e. normal day 5 blastocysts
`that had been exposed to endogenous estrogen action, were used for the transfer
`experiments. EGF initiated implantation of these embryos
`(group 1, Table III)
`but only 26% of those transferred implanted. The growth factor also initiated
`implantation of normal day 5 blastocysts when the recipient was injected with
`the anti—estrogen.
`ICI—182 was given on day 4 of pregnancy to the donor of the
`embryos with the thought of
`inhibiting the effect of endogenous estrogen and
`thus possibly preventing the implantation initiating action of EGF. However,
`the
`growth factor was at
`least as effective at
`inducing implantation of
`these
`embryos as those from the control animals. This suggests that the effect of ICI-
`182 does not mimic the effect of hypophysectomy in producing delayed implanting
`embryos.
`
`ICI~182 effectively
`the purely antagonistic anti-estrogen,
`In summary,
`prevents
`the effect of
`endogenous and exogenous estradiol
`for
`initiating
`implantation but even at doses 20 times those effective against the steroid it
`does not inhibit the effect of EGF for the same response.
`
`Acknowledgments
`
`from the National
`(ES03950)
`This research was supported in part by a grant
`(HD02528)
`to the
`Institute of Environmental Health Sciences, and a core grant
`R.L. Smith Research Center from the National Institute of Child Health and Human
`Development. We
`thank Drs. B. Vose and A.E. Wakeling (ICI Pharm)
`for anti—
`estrogen.
`
`w
`
`l.
`
`2.
`
`3.
`
`5.
`
`\OGJQOX
`
`in Handbook of PhysiologyI Section 7,Vol III Part 2, R.O.
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`AstraZeneca Exhibit 2161 p. 5
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`Vol. 53, No. 21, 1993
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`AstraZeneca Exhibit 2161 p. 6
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