`estrous behavior and energy balance in Syrian hamsters
`
`GEORGE N. WADE, Jr BRADLEY POWERS,
`JEFFREY I). BLAUSTEIN, AND DEBORAH E. GREEN
`Department of Psychologv and Neuroscience and Behooior Program,
`University of Massachusetts, Amherst, Massaehusetm 01063
`
`Wade, George N., J. Bradley Powers, Jeffrey I).
`Blaustein, and Deborah E. Green. 10] 182,780 antagonizes
`the effects of estradiol on estrous behavior and, energy balance
`in Syrian hamsters. Am. J, Physiol. 265 (Reguhtmzv Integrative
`Comp. Physiol. 34): R1399-R1403, 1993.-Three experiments
`examined the effects of 101 182,780, a steroidal “pure” armies—
`trogen that is thought to be active peripherally but not in the
`brain when given systemically, on energy balance, estrom be-
`havior, and in vivo coll nuclear binding of Pillow-Mini in Syr-
`ian hamsters. Pretreatment with lCl 182,780 reduced in vivo
`uptake of [amestz‘adiol in uterus but not in pooled hypothala-
`mue~preoptie area. mafieetomized Syrian hamsters were
`treated with estradiol benzoate (BB, 5 ngg'day}, ICI 182380 (250
`gig/day}, or both EB and 101 182380 for 4 wk. Estradiol treat-
`ment caused significant decreases in food intake, body weight
`and fat content, and linear growth. Given alone, ICl 182.?80
`had no effect on these measures. When they were given concur-
`rently, 101 182,780 attenuated the effects of estradiol on body
`weight, growth, and fat content but not on food intake. ‘I‘reat~
`ment with 101 182,780 significantly diminished estrous behav-
`ior induced with either EB plus progesterone or with EB alone.
`These findings support the hypothesis that, in addition to its
`actions in the brain, estradiol acts peripherally to modulate
`estmua behavior and energy balance.
`hotly weight; body composition; uterus; food intake; estrogen
`receptors
`
`affect other social behaviors (21), but the possibility of
`peripheral sites of action has not been tested. Second, it
`has been suggested that estradiol can act peripherally to
`alter lipid metabolism and energy balance in hamsters
`(3), but the evidence for this notion is rather indirect.
`Third, hamsters and rate differ in some of their re—
`sponses to antiestrogens. The older noneteroidal antige—
`trogens such as MEIR-25, {II-628, and tamoxifen inhibit
`steroid-induced estrous behavior in both species {13, 14,
`17, 25). However, for regulation of energy balance these
`compounds are full estrogen agoniete in rats, whereas
`they act as antagonists in hameters (24, 25L Thus the
`newer, steroidal antiestrogens such as ICI 182380 may
`have different effects in rats and hamsters.
`
`METHOI’IS
`
`Animals and Housing
`
`Adult, female Syrian hamsters (Mesocricetus auratus; initial
`body wt 90—110 g) of the Lak:LVG strain were obtained from
`Charleg River Breeding Laboratories (Wilmington, MA). Ham—
`sters were housed in wire—bottom stainless steel cages (17.5 x
`17.5 x 25 cm) and given tap water and Purina Laboratory
`Rodent Chow (no. 5001} ad libitum. Food pellets wore placed on
`the cage floor. A 14:18-11 lightvtiark cycle was maintained (lights
`on at Will”) it}, and room temperature was kept at 22 i 2" C. After
`1 wk of acclimation to the laboratory. animals were ovarieoto—
`mined via bilateral flank incisions under pentubarbital smiium
`anesthesia {80 mgfkg; Sigma Chemical, St. Mule, MO}.
`
`Procedures
`
`1r IS CLEAR THAT ommm steroids can act directly in
`the brain to affect a wide variety of behaviors and physi—
`ological functione, including social behaviors, regulatory
`behaviors, and energy balance {2, 4, 21, 23, 26}. For
`example, appropriately placed intracerebral implants of
`estradiol facilitate estrous behavior, decrease food in-
`In vino binding of PHjestmdiol. Three weeks after ovarian-
`take, or stimulate voluntary exercise in ovariectomized
`tomy, hamsters were given three daily injections of sesame oil
`in = 6) or 250 ,ug ICI 182,780 in = 6). One hour after the third
`rats (6. “1.8, 27}; lesions of these neural loci prevent the
`injection, animals were injeeted intraperitoneally with 60 nCi
`respective behavioral changes in response to systemic
`[3H]estradioi (Sp act 103 Ci/mmol, New England Nuclear, Bos-
`estradiol treatment (7. 9, 12).
`‘wn. MA)‘ One hour after injection of [aHleetradioL hamsters
`In addition to theee central actione, several lines of
`were anesthetized with pentobarbital sodium (50 mg); a blood
`evidence support the hypothesis that estradiol can act
`eample was taken via cardiac puncture with a heparinized sy-
`on nonneural peripheral tissues to affect behaviors and
`ringe and then centrifuged. Hamsters were then perfused with
`energy balance in rats (22, 23, 26}. The fact that the
`cold saline {0.15 M). The hypothalamus-prawns area and
`antieetrogen ICI 182,780 attenuates the effects of estra~
`uterus were rapidly dissented. Tissues were homogenizeci, and a
`dioi on energy balance and estrous behavior in ovarian-
`cell nuclear fraction was purified by a modification (8) of the
`tomized rats is consistent with the existence of periph—
`method of Zigmond and McEwen {313). Radioactivity was ex
`trashed from the purified cell nuclei with 3 X 4 ml toluene—based
`eral sites of action (22). TLC-1182380 differs from other
`scintillation fluid. A lOO-nl aliquot of plasma was transferred to
`antiestrogens in that it is highly potent peripherally but
`a ecintillation vial containing 12 ml of scintillation fluicl and
`it does not; appear to be active in the brain when it is
`shaken vigorously. Radioactivity was counted at an efficiency of
`administered systemically (22, 28, ‘29).
`~45%, and counts were corrected for quenching by automatic
`The idea that estradiol acts both centrally and periph«
`external standardization. Protein in cell nuclear samples was
`orally to affect behaviors and energy balance has not
`precipitated with ethanol. dissolved in 0.3 N KOH, and esti—
`been explored in species other than rats. The present
`mated by the method of Bradford (5). Tianne cell nuclear con-
`experiments use ICI 182,780 to investigate this possibil»
`centrations of radioactivity are expressed as tissue to plasma
`ity in Syrian hamsters, Hamsters are of interent for
`retina (La, disintegratione per minute per milligram tissue pro-
`several reasons. First, it is known that estradiol can act
`tein divided by disintegrations per minute per microliter blood
`directly in the brain to facilitate sexual receptivity and
`plasma).
`0363-6119193 $200 Copyright © l993 the American Physiological Society
`
`R1399
`
`AstraZeneca Exhibit 2109 p. 1
`InnoPharma Licensing LLC V. AstraZeneca AB IPR2017-00900
`
`
`
`R1400
`
`ANTIESTRDGEN EFFECTS IN HAMSTERS
`
`Uterus
`
`Brain
`
`J:- OO
`
`to OO
`
`NOC)
`
`Ec:
`
`UEU
`
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`2G.\
`OJ
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`
`3V
`
`Energy balance. Three weeks after ovariectomy, baseline food
`intake (pouching and spillage accounted for) and body Weight
`were measured to the nearest 0.1 g. After 1 wk of data collection,
`animals were divided into four groups matched for baseline food
`intake and body weight. The groups were given daily subcuta-
`neous injections of 0.1 ml sesame oil vehicle containing 2.5%
`ethanol (n = 11), 5 pg estradiol benzoate (EB, n = 10), 250 ,ug
`101181780 (n. = 11), or 5 pg EB plus 250 fig ICI 182,780 in =
`10) for 4 wk. Body weight and food intake were measured twice
`a week. On the first and last days of injections, hamsters were
`anesthetized with pentobarbital sodium (80 mgf'kg body wt),
`and nsso-anal length was measured to the nearest millimeter
`with calipers while the animals were stretched with a constant
`IOU-g weight (24). The difference between the two lengths is an
`index of linear growth during hormone treatment.
`At the end of the experiment all animals were killed with an
`overdose of pentobarbital sodium (50 mg). Parametrial and ret-
`roperitoneal fat pads and uteri were removed and weighed. Evia—
`cerated carcasses were dried to a constant weight at 70° C, and
`carcass lipid was estimated from carcass water content. In ham-
`sters, percent carcass water and percent carcass lipid are highly
`correlated (r = 0.98; %lipid = —1.3‘2 X %water + 96.54; n = 289;
`Wade, unpublished data). Fat-free dry weight was calculated as
`the total evisccrated carcass weight less water and estimated
`lipid content.
`ESEP‘OELS behavior. The animals that were treated with EB,
`1C1182,780, or EB plus ICI 182,780 (above) were tested for
`estrous behavior twice, once with and once without progeste-
`rone treatment. On the 3rd day of estrogen and/or antiestrogen
`treatment, all animals were given a subcutaneous injection of
`200 pg progesterone in 0.1 ml sesame oil at 0900 h and tested for
`estrous behavior 5 to 6 h later. The second test, without. pro-
`gesterone, took place on the 23rd day of estrogen and/or anti-
`estrogen treatment. For both tests, females were adapted to a
`small Plexiglas arena (30 X 36 X 30 cm) for 5 min. Sexual
`receptivity tests were begun by introducing a sexually active
`male to the arena. In addition, the female’s flanks and perigenie
`tal region were continuously stimulated using an eyelid brush.
`Tests lasted for 180 s; the latency to display lordosis and the
`total time that the lordosis posture was maintained were re-
`corded. Tests were conducted 6—8 h after lights—on.
`
`Data A nalyses
`
`
`
`lCl
`Oil
`iCI
`Oil
`Fig. 1. Effects of ICI 182,780 (ICI) on in vivo uptake of [3H]estradiol by
`cell nuclei in uterus and hypothalamus-preoptic area in ovariectomized
`hamsters. Animals were treated with sesame oil vehicle (0.1 ml) or ICI
`(250 pg) 48, 24, and 1 h before injection of [3H]estradiol. Data are
`expressed as tissue to plasma ratios, that is, disintegrations per minute
`per milligram cell nuclear protein divided by disintegrations per minute
`per microliter plasma. * P <: 0.05 vs oil-treated group.
`
`Food intake (gfday)
`
`nl
`li
`
`_,
`x
`I
`«riff:—
`‘97?“—
`">ka 8
`
`1':1%:\
`1’
`
`on
`(£10
`EB+lCl
`
`41
`
`I
`
`l
`
`|
`
`Body weight change (9)
`
`20
`
`Data were analyzed by t tests and by one- or two -way analyses
`of variance followed by NewmamKeuls post hoc tests where
`appropriate. Differences were considered statistically signifi»
`cant when P < 0.05 for two tails. Data are expressed as means
`t SE.
`
`Doys oflreolmenl
`Fig. 2. Body weight gain and food intake of ovariectomized hamsters
`treated with sesame oil vehicle (0.1 ml), estradiol benzoate (EB, 5 ng/
`day), ICI (250 rig/day), or EB plus ICI for 4 wk. (Initial mean group body
`weights 13424359 3,.)
`
`RESULTS
`
`In Vivo Binding of [3HjEstrodiol
`Pretreatment with ICI 182,780 significantly reduced in
`vivo cell nuclear binding of [3H]estradiol in uterus but
`not in pooled hypothalamus-preoptic area (Fig. 1), simi-
`lar to findings with rats (22). However, uterine [3H]-
`estradiol binding was reduced by only ~50% in hamsters
`compared with ~90% in rats (22).
`
`Energy Balance
`EB treatment significantly reduced food intake and
`body weight gain (Fig. 2). ICI 182,780, given alone, had
`no effect on body weight, but it significantly attenuated
`the weight—reducing actions of EB. Given alone or in
`combination with EB, ICI 182,780 had no effect on food
`intake (Fig. 2).
`
`Treatment with EB caused significant reductions in
`carcass water and lipid but not in fat-free dry weight (Fig.
`3). Once again, ICI 182,780 alone had no effect, but it
`antagonized the effects of EB on carcass lipid content.
`The changes in body fat content were reflected in the
`weights of individual fat pads (Fig. 4). EB also caused a
`significant decrease in linear growth (change in nasoanal
`length) that was prevented by concurrent treatment with
`ICI 182,780 (Fig. 4). Administration of ICI 182,780 alone
`did not affect growth.
`EB treatment caused a significant increase in uterine
`weight. As in other species (22, 28, 29), 1C1 182.780 had
`no uterotrophic effect, and at this d039, the antiestrogen
`partially antagonized the effect of EB on uterine weight
`(Fig. 4).
`
`AstraZeneca Exhibit 2109 p. 2
`
`
`
`Carcass water (9)
`
`Carcass lipid (g)
`
`For-free dry wt. lg)
`
`Parametric! WAT (g)
`
`Retroperit. WAT (g)
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`R1401
`
`a
`
`b
`
`l8
`
`12
`6
`
`24 c
`
`Oil
`
`EB lCl EB+
`ICI
`
`Oil
`
`EB
`
`ICI EB+
`|C|
`
`Oil
`
`EB ICl EB+
`ICl
`
`Fig. 3. Terminal carcass composition of ovariectomized hamsters
`treated with sesame oil vehicle [0.1 m1), EB (5 pig/day}, ICI (.250 ug/
`day), or EB plus ICI for 4 Wk. Bars with different letters are signifi-
`cantly different (P < 0.05).
`
`Estrous Behavior
`
`Treatment with ICI 182,780. given alone or in combi-
`nation with progesterone, did not induce any signs of
`sexual receptivity in ovariectomized hamsters (data not
`shown). However, ICI 182,780 did inhibit estrous behav-
`ior induced by treatment with either EB plus progeste—
`rone or EB alone. ICI 182.780 significantly increased
`lordosis latency and decreased lordosis duration in ham-
`sters treated with EB for 2 days followed by progesterone
`(Fig. 5, top). In hamsters given EB for 23 days, concur-
`rent
`treatment with ICI 182,780
`significantly de-
`creased lordosis duration but did not affect latency (Fig.
`5, bottom).
`
`DISCUSSION
`
`These findings confirm a number of the unusual prop-
`erties of ICI 182,780. As in other species (22, 28, 29),
`ICI 182,780 appears to be a pure antiestrogen in Syrian
`hamsters. It at. least partially antagonized the actions of
`estradiol on body weight, body composition,
`linear
`growth, estrous behavior, and uterine weight without
`having any agonistic (estrogenic) effects when given by
`itself to ovariectomized animals. The present results also
`support the conjecture that ICI 182,780 does not act di—
`rectly in the brain. As in rats (22), pretreatment with
`radioinert ICI 182,780, significantly reduced in vivo cell
`nuclear binding of [3H]estradiol in uterus but not in
`pooled hypothalamus-preoptic area. Taken together,
`these two findings suggest that ICI 182,780 can he used as
`an experimental tool to dissociate central and peripheral
`actions of estradiol.
`The fact that ICI 182,780 attenuated the effects of es—
`tradiol treatment on body weight and fat content is con-
`sistent with our assertion (23, 26) that ovarian steroids
`act both centrally and peripherally to affect energy bal~
`once. Several lines of work support the idea of distinct
`central and peripheral sites of estrogen action on energy
`balance in rats (22, 23, 26), but this appears to be the first
`evidence for this possibility in hamsters.
`The fact that treatment with ICI 182,780 completely
`blocked the effects of estradiol on linear growth (change
`in case-anal length) may indicate that estrogen effects on
`growth are predominantly due to nonneural actions of the
`steroid in hamsters. On the other hand, treatment with
`ICI 182,780 did not attenuate the suppressive effects of
`estradiol on food intake in hamsters. Thus it is likely that
`estradiol action in the brain is sufficient to decrease food
`
`1.00
`
`.75
`
`.50 .25
`
`Oil
`
`EB
`
`tCI on
`ICI
`
`on
`
`E8
`
`lCI EB+
`ICI
`
`Growth (mm)
`
`o
`
`a
`
`a
`
`b
`
`B
`o
`
`4
`
`2
`
`.500
`.375
`
`.230
`
`.125
`
`"on EB.
`
`to! as:
`lCI
`
`Uterus (g!
`b
`
`
`
`Oil
`
`to
`
`IE! EB+
`lCl
`
`Fig. 4. Terminal weights of parametric! and retroperitoneal white adi—
`pose. tissue (WAT) and uterus and change in naso—ana] length (growth)
`of ovariectomized hamsters treated with sesame oil vehicle (0.1 ml), EB
`(5 rag/day), ICI (250 rig/day), or EB plus ICI for 4 wk. Ears with differ-
`ent letters are significantly different (P < 0.05).
`
`Estradiol-I-progesterone:
`a
`Latency (sec)
`Duration (sec)
`
`60
`
`40
`
`20
`
`*
`
`120
`
`80
`
`40
`
`EB
`
`EB+
`ICI
`
`EB
`
`EB+
`ICI
`
`Estradiol alone:
`Latency [sec]
`
`60
`
`40
`
`20
`
`Duration (sec)
`
`
`
`so
`
`40
`
`EB
`
`EB
`
`EB+
`EB+
`ICI
`ICI
`Fig. 5. Effects of ICI on estrous behavior in ovariectomized hamsters.
`Top: animals were injected with BB [5 pg) or EB plus ICI (250 mg) for
`2 days followed by 200 pg progesterone on 3rd day, 4—5 h before testing.
`Bottom: animals were injected with EB or EB plus ICI for 23 days before
`testing. * P < 0.05 vs. EB-treated group.
`
`intake and that no peripheral actions are required. How-
`ever, we cannot exclude the possibility that a higher dose
`of ICI 182,780 than that used in this study would be
`sufficient to antagonize the effects of estradiol on food
`intake in hamsters.
`
`AstraZeneca Exhibit 2109 p. 3
`
`
`
`R1402
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`These findings in hamsters contrast with those in rats
`where ICI 182,780 treatment actually potentiated the es-
`tradiol-induced decreases in food intake (22). We sug-
`gested that in rats this action of ICI 182,780 could be due
`to the induction of a “pharmacological hysterectomy,”
`because surgical hysterectomy enhances behavioral re-
`sponsiveness to estradiol in this species (1, 20). In con~
`trast, surgical hysterectomy does not potentiate estradiol-
`induced sexual receptivity in hamsters (19),
`just as
`ICI 182,780 treatment does not potentiate the estradiol—
`induced decrease in food intake. Therefore, the difference
`between rats and hamsters in the effects of ICI 182,780
`on food intake may be a reflection of the species differ-
`ence in the actions of the uterus on behavioral respon-
`siveness to estradiol.
`
`ICI 182,780 attenuated the induction of estrous behav-
`ior, either by sequential treatments with estradiol and
`progesterone or by prolonged (23 days) treatment with
`estradiol alone. Thus, in hamsters, as in rats (22), estra-
`diol appears to act peripherally, as well as centrally'(2, 4,
`21), to facilitate estrous behavior. We have suggested
`that in rats, ICI 182,780 could attenuate sexual receptiv-
`ity at least in part by blocking the effects of estradiol
`on peripheral sensory fields that are important for es—
`trous responsiveness (10, 11). Similar work examining
`the effects of ovarian steroids on peripheral sensory
`fields has not yet been done in hamsters, but it is known
`that
`somatosensory cues
`from the perineal
`region
`play a significant role in hamster copulatory behavior
`(15, 16).
`are possible,
`comparisons
`that
`extent
`To the
`ICI 182,780 may be more effective at inhibiting estrous
`behavior in hamsters than in rats (22). If this is in fact
`the case, the uterus might play a role in this species dif-
`ference, because surgical hysterectomy potentiates the
`effects of estradiol on sexual receptivity in rats but not
`in hamsters (1, 19). Thus in rats ICI 182,780 could con-
`currently inhibit estrous behavior
`(perhaps by inhi—
`biting an action of estradiol on peripheral sensory
`fields) and facilitate estrous behavior (via a pharmaco-
`logical hysterectomy facilitating estrogen action in
`the brain). According to this hypothesis, only the inhibi—
`Lory actions would be evident in hamsters.
`It appears as though the dose of ICI 182,780 we have
`used (250 Jug/day) is more effective in reducing uterine
`weight and in vivo cell nuclear uptake of {3H]estradiol in
`rats than in hamsters (22). At this time it is not clear
`whether this effect is due to 1) species differences in
`101182.780 absorption, delivery, or clearance; 2) dif—
`ferences in affinity for estrogen receptors; or 3) some
`other factorls). Differences in the EB doses that were
`used (2 ug in rats, 5 ug in hamsters) could explain the
`differences in the inhibition of uterine weight but can-
`not account for the differences in inhibition of [3H]es-
`tradiol binding. Whatever the basis for this species dif—
`ference in ICI 182,780 potency in the uterus, the impor-
`tant point remains that
`in both rats and hamsters
`treatment with ICI 182,780 attenuates the effects of es—
`tradiol on energy balance and on estrous behavior with—
`out interfering with neural estrogen binding. Therefore,
`these results support the hypothesis that estradiol affects
`
`energy balance and estrous behavior via both neural and
`nonneural sites of action.
`
`We are grateful to Jay Alexander, Robin Lempicki, and Joanne Tur-
`cotte for their expert technical assistance and to Alan E. Wakeling of
`Zeneca (ICI) Pharmaceuticals for the gift of ICI 182,780.
`This work was supported by Research Grants N8-10873. DK-32976,
`and NS-19327, by Research Scientist Award MH-OOSEI, and by Re-
`search Scientist Development Award NIH-00885 from the National Iu-
`stitutes of Health.
`Address reprint requests to G. N. “fade, Dept. of Psychology, Univ.
`of Massachusetts, Amherst, MA 01003.
`Received 8 March 1993; accepted in final form 5 May 1993.
`
`REFERENCES
`
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`12. Law, 'I‘., and W. Meagher. Hypothalarnic lesions and sexual
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`13. Moisel, R. L., G. P. Dohanich, B. S. McEwen, and D. W.
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`AstraZeneca Exhibit 2109 p. 4
`
`
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`R1403
`
`18.
`
`19.
`
`20.
`
`21.
`
`22.
`
`23.
`
`24.
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