ICI 182,780 antagonizes the effects of estradiol on
`estrous behavior and energy balance in Syrian hamsters
`
`GEQRGE N. WADE, J. BRADLEY POWERS;
`JEFFREY I). BLAUSTEIN, AND DEBORAH E. GREEN
`Bepartment of Psychology and Neuroscience and Behuwbr Program,
`University of Maamchusetts, Amherst. llrfassashusettr 0100!?
`
`Wade, George N., J. Bradley Powers, Jeffrey D.
`Blaustein. and Deborah E. Green. I01 182,780 antagonizea
`the effects of estradiol on estrous behavior and energy balance
`in Syrian harnabors. Am. J. Physiol. 265 {Regulatory Integrative
`Comp. Physioi. 34): R1399-R1403, 1993.~—Three experiments
`examined the effects of ICI 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, ealrowz be-
`havior, and in viva cell nuclear Binding of [3H}estradiol in Syr-
`ian hamsters. Pretreatment with ICE 182,3?-0 reduced in viva
`uptake of [31~l}estz*adiol in uterus but not in pooled hypothala-
`mua—preoptic area. Gvarieotumized Syrian hantsters. were
`treated with estradiol ban:-aoate (E33, 5 ;xg,"d£!.y}, }CI 182,780 (250
`ug/day}. or both EB and R31 132,780 for 4 wk. Eatradiol treat-
`ment caused significant decreases in food intake, body weight
`and fat content, and linear growth. Given alone, IC1 182,780
`had no effect on these measures. When they were given concur-
`rently, ICI 132,’? 80'a1;tenuated the effects of estradiol on body
`weight, growth, and fat content but not on food intake. ‘Treat-
`ment with ICI 182,’/80 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, ea:-slradiol acts peripherally to modulate
`estrous behavior and energy balance.
`body; 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 Quggested that es-tradiol can act peripherally to
`alter lipid metabolism and energy balance in hamsters
`(3), but theevidence for this notion is rather indirect.
`Third, hamsters and rats differ in some of their re-
`sponses to antiestrogens. The older nonsteroidal amiss-
`trogens such as MER-25, (31-1328, and tamoxifen inhibit
`5teroi<i—induced estrous behavior in both species (13, 14,
`17, 25). However, for regulation of energy balance these
`compounds are full estrogen agonists in rats, whereas
`they act as antagonists in hamsters (24, 25}. Thus the
`newer, steroidal antiestrogens such as ICI1S2,7BO may
`have different effects, in rats and hamsters.
`
`METH ODS
`
`A rrimals and Housing
`Adult, female Syrian hamsters (Mesocricetus aumtus; initial
`body wt 90-110 3) of the Lal<:LVG strain were obtained from
`Charles River Breeding Laboratorizrs (Wilmington, MA). Ham-
`sters were housed in wit.-e—bottom stainless steel cages (1725 X
`17.5 X 25 cm) and given tap water and Purina Laboratory
`Rodent Chow (no. 5001} ad lihitum. Food pellets. were placed on
`the cage floor. A 14:18-ll light-dark cycle was maintained (lights
`on at 6700 h}, and room temparalure was kept at 22 i 2" C. After
`1 wk of acclimation to the laboratory, animals were ovariecto
`mizzexzi via bilateral flank incisions under pentobaxbital sodium
`anesthesia [S-G mgfkg; Sigma Chemical, St. Louis, MO).
`
`Procedures
`
`rr IS CLEAR THAT omnrm steroids can act directly in
`the brain to affect a wide variety of behaviors and physi-
`ological functions, including social behaviors, regulatory
`behavicsrs, and energy balance (2, 4, 21. 23. 26). For
`example, appropriately placed intracerebral implants of
`estradiel facilitate estrous behavior, decrease food in-
`In viva biriding of Pfflestradioi. Three weeks after ovarian-
`take, or stimulate voluntary exercise in ovariectomized
`tomy, hamsters were given. three daiily injections of sesame oil
`(n == 6) or 250 pg ICI 182.7813 (n = 63. One hour after the third
`rats (6, 18, 27); lesions of these neural loci prevent; the
`injection, animals were injected intraperitoneally with 61] ,uCi
`respective behavioral changes in response to systemic
`["‘H]estradiol (sp act 103 Ci/mmnl, New England Nuclear, Bos-
`estradiol treatment (7, 9, 12).
`ton. MA). One hour after injection of [*‘H}e3M-adiol, hamsters
`In addition to these central actions, several lines of
`were anesthetized with pentoharbital sodium (50 mg); a blood
`evidence support the hypothesis that estradiul can act
`sample was taken via cardiac puncture with a heparinized sy-
`on nonneura] 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 hypothalamuss-preoptic area and
`antiastrogen ICI 182330 attenuates the effects of estra~
`uterus were rapidly dissected. Tissues were homogenized, and a
`diol on energy balance and estrous behavior in mariac-
`cell nuclear fraction was purified by a modification {8} of the
`tcmtiized rats is consistent with the existence of periph-
`method of Zigmond and Mclilwen (30). Radioactivity was ex—
`treated from the purified cell nuclei with 3 >4 é ml toluene—based
`eral sites of action (22). ICI 182,780 differs from other
`xintillation fluid. A 1013121 aliquot of plasma was transferred to
`antiestrogexxs in that it is highly potent peripherally but
`a scintillation vial containing 12 ml of scimillation fluid and
`it does not appear to be active in the brain when it is
`shalom vigorously. Radioactivity was counted at an efficiency of
`administered systaemically (22, 28, 29).
`~-15%, and counts were corrected for quelrchizxg hy autamntir:
`The idea that eat:-adiol acts both centrally and periph~
`external standardimtion. Protein in cell nuclear samples was
`erally to affect bahaviors and energy balance has not
`precipitated with ethanol, dissolved in 0.3 N K011, and esti-
`been explored in species other than rats. The present
`mated 1:351 the method of Bradford (5). Tieame 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 interest for
`ratios (Lel, disintegrationa per minute per milligram tissue pro-
`several ‘reasons. First, it is known that est:-adiol can act
`tein divided by disintegratiorxa per minute per microliter blood
`directly in the brain to facilitate sexual receptivity and
`plasma).
`0363-6119,-‘Q3 $2.00 Copyright © L993 the American Physiological Society
`
`R1399
`
`Astrazeneca Ex. 2109 p. 1
`Mylan Pharms. Inc. V. Astrazeneca AB IPR2016-01316
`
`

`
`R1400
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`Uterus
`
`Brain
`
`*
`
`8
`6
`
`4
`
`2
`
`400
`
`CI
`E
`3 300
`
`E g
`
`3g
`
`! 200
`
`l00
`
`ICI
`Oil
`10
`Oil
`Fig. 1. Effects of ICI 182,780 (ICI) on in vivo uptake of [3H]eatradiol by
`cell nuclei in uterus and hypothalamus-preoptic area in ovariectomized
`hamsters. Animals were treated with sesame oil vehicle (0.1 ml) or D3!
`(250 pg) 48, 24, and 1 h before injection of [3H]estradio1_ 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 <1 0.05 vs C-il—treated group.
`
`_:
`
`-
`
`“CH
`
`EB.‘-[Cl
`
`Food intake (g/day)
`
`A
`nl
`
`li:"93.‘\‘*-e
`‘A . P,
`
`L
`
`5 4
`
`Body weight change (9)
`
`20
`
` 0
`
`W
`
`7
`
`23
`
`21
`14
`Days of freolmem
`Fig. 2. Body weight gain and food intake of ovsriectoiniaed hamsters
`treated with sesame oil vehicle (0.1 ml), estradiol lienzoate (E13, 5 pg/
`day}, ICI (250 pg/day), or EB plus ICI for 4 wk. (Initial mean group body
`weights 13-1.2—135.9 g.)
`
`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 berizoate (EB, n = 10), 250 pg
`ICI 182,780 (11. = 11), or 5 pg EB plus 250 pg 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 pentobarhital sodium (80 mg/kg body wt),
`and naso-anal length was measured to the nearest millimeter
`with calipers while the anirnais were stretched with a constant
`100-5; 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: ‘Kilipid = -1.32 X ‘léwater + 96.54; n = 289;
`Wade, unpublished data). Fat-free dry weight was calculated as
`the total eviscerated carcass weight less water and estimated
`lipid content.
`Estrous behavior. The animals that were treated with EB,
`ICI 182,780, 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 09130 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 perigeni—
`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 Analyses
`
`Data were analyzed by t tests and by one- or two -way analyses
`of variance followed by Newman~Keuls 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.
`
`RESULTS
`
`In Viuo Binding of [3H]Estradz'ol
`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). Aclrninistratinn 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 dose, the hntiestrogen
`partially antagonized the effect of EB on uterine weight
`(Fig. 4).
`
`Astrazeneca Ex. 2109 p. 2
`
`

`
`24 c
`
`Oil
`
`Oil
`
`EB
`
`EB |Cl EB?!-
`ICI EE+
`EB ICI EB+
`IC‘.
`ICI
`ICI
`Fig. 3. Terminal carcass composition of ovariectomized hamsters
`treated with sesame oil vehicle [(1.1 ml), EB [5 pig/clay}, ICI (250 ug/
`day), or EB plus ICI for 4 wk. Bars with different letters are signifi-
`cantly different (P -21 0.05).
`
`ca rcass water lg)
`
`Carcass lipid lg)
`
`Fat-free dry wt. lg)
`
`Purametriol WAT lgl
`
`R9lg0P3|'"- WAT 191
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`R1401
`
`°
`
`b
`
`l8
`
`1?
`ti
`
`
`
`Oil
`
`EB
`
`ICI
`
`EE-+
`1::
`
`on
`
`EB
`
`lC| EM
`non
`
`Oil
`
`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.
`ICT 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),
`ICI182,78O 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
`redioinert ICI 182,780, significantly reduced in vivo cell
`nuclear binding of [31-Ilestradiol 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 eetradiol.
`The fact that ICI 182,780 attenuated the effects of as-
`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~
`ance. 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 eetradiol on linear growth (change
`in naso-anal length) may indicate that estrogen effects on
`growth are predominantly due to nonneural actions of the
`steroid in harnsters. 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
`
`Growth lmrnl
`
`(1
`
`O
`
`G
`
`b
`
`8
`o
`
`4
`2
`
`.500
`.375
`
`.230
`.125
`
`"on re
`
`Uterus (91
`b
`
`
`
`on
`
`to 1:: 53+
`lcl as?
`lCl
`ICI
`Fig. 4. Terminal weights of parametrial and retroperitoneal White adi~
`pose tissue (WAT) and uterus and change in naso—anal length (growth)
`of ovariectoinizecf hamsters treated with sesame oil vehicle (0.1 ml), EB
`(5 pg/day], ICI (250 pg/day}, or EB plus ICI for 4 wk. Ears with differ-
`ent letters are significantly different (P < 0.05).
`
`Estrodiol-rprogesferone:
`1
`Latency (sec)
`Duration (sec)
`
`60
`
`40
`
`20,
`
`"‘
`
`120
`
`80
`
`40
`
`EB
`
`EB+
`ICI
`
`EB
`
`EB+
`ICI
`
`Esfradiol alone:
`
`Latency (sec)
`
`Duration (sec)
`
`60
`
`40
`
`20
`
`It
`
`120
`
`80
`
`40
`
`EB
`
`EB
`
`EB+
`EB+e
`ICI
`ICI
`Fig. 5. Effects of ICI on estrous behavior in ovarieetomized hamsters.
`Top: animals were injected with EB [5 gig) or EB plus ICI (250 fig} for
`2 days followed by 200 fig progesterone on 3rd day, 4-5 h before testing.
`Bottom: animals were injected with EB or EB plus ICI for23 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 Ex. 2109 p. 3
`
`

`
`R1402
`
`ANTIESTROGEN EFFECTS IN I-IAMSTERS
`
`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
`ICI182,780 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 (2 2), 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
`thebrain). According to this hypothesis, only the inhibi-
`tory 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
`ICI182,780 absorption, delivery, or clearance; 2) dif-
`ferences in affinity for estrogen receptors; or 3) some
`other factor(s). Differences in the EB doses that were
`used (2 gig in rats, 5 ng 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 NS-10873. DK-32976.
`and NS-19327, by Research Scientist Award MH-00321, and by Re-
`search Scientist Development Award MH—00885 from the National In-
`stitutes of Health.
`Address reprint requests to G. N. Vifade, Dept. of Psychology, Univ.
`of Massachusetts, Amherst. MA 01003.
`Received 8 March 1993; accepted in final form 5 May 1993.
`
`REFERENCES
`
`1. Ahdieh, H. B., and G. N. Wade. Effects of ‘hysterectomy on
`sexual receptivity, food intake, running wheel activity, and hypo-
`thalamic estrogen and progestin receptors in rats. J. Comp.
`Physioi. Psychol 96: 886-892, 1982.
`2. Barfield, R. J., B. S. Rubin, J. H. Glaser, and P. G. Davis.
`Sites of action of ovarian hormones in the regulation of oestrous
`responsiveness in rats. In: Hormones and Behaviour in Higher
`Vertebrates. edited by J. Balthazart. E. Prove. and R. Gilles. Ber-
`lin: Springer-Verlag, 1983, p. 2-17.
`3. Bhatia, A. J ., and G. N. Wade. Effects of pregnancy and ovar-
`ian steroids on fatty acid synthesis and uptake in Syrian hamsters.
`Am. J. Physiol. 260 (Regulatory Integrative Comp. Physiol. 29}:
`R153-R158, 1991.
`4. Blaustein, J. B., and D. H. Olster. Gunadal steroid hormone
`receptors and social behaviors. In: Advances in Comparative and
`Environmental Physiology, edited by J. Balthazart. Berlin:
`Springer-Verlag, 1989, vol. 3, p. 31-104.
`5. Bradford, M. M. A rapid and sensitive method for quantifica-
`lzionof micrograrn quantities of protein utilizing the principle of
`protein—dye binding. Anal. Biochem. 72: 248-254, 19376.
`6. Butera, P. 0., and R. J. Beikirch. Central
`implants of
`diluted estradioi: independent effects on ingestive and reproduc-
`tive behaviors of ovariectomized rats. Brain Res. 491: 268-273,
`1989.
`
`7. Butera, P. C., D. M. Willard, and S. A. Raymond. Effects of
`I-‘VN lesions on the responsiveness of female rats to estradiol.
`Brain Res. 516: 304-310. 1982.
`8. Gentry, R. T.. G. N. ‘Wade. and E. J. ROY. Individual differ-
`ences in estracliol-induced behaviors and in neural “H-estradiol
`uptake in rats. Physiol. Behcu. 17: 195-200, 1976.
`9. Kennedy, G. C. I-Iypothalamic control of the endocrine and be-
`havioural changes associated with oestrus in the rat. J. Physiol.
`Land. 172: 383-392. 1964.
`10. Komisaruk, B. R., N. T. Adler, and J. Hutchinson. Genital
`sensory field: enlargement by estrogen treatment in female rats.
`Science Wash. DC 178: 12954298, 1972.
`11. Kow, L.~M., and D. W. Pfaff. Effects of estrogen treat-
`ment on the size of receptive field and response threshold of
`pudenclal nerve in the female rat. Neuroendocrinulugy 13: 299-313,
`1973.
`12. Law, ’I'., and W. Meagher. Hypothalamic lesions and sexual
`behavior in the female rat. Science Wash. DC 128: 1626-1627,
`1953.
`13. Meisel, R. L., G. P. Dohanich, B. S. McEWen, and D. W.
`Pfaff. Antagonism of sexual behavior in female rats by ventra-
`lnedial hypothalamic implants of antieotmgen. Neu.roendocrin.o1-
`ogy 45: 20l—207, 1987.
`14. Morin, L. P.. J. B. Powers. and M. White. Effects of the
`antiestrogens, MER-25 and 131-628, on rat and hamster lurdoais.
`Harm. Behcw. T: 233-291, 1976.
`15. Noble, R. G. The sexual responses of the female hamster; a
`descriptive analysis. Ph.y5'ioL Bolton. 23: 1001-1004, 1979.
`16. Noble, R. G. Sex responses of the female hamster: effects on male
`performance. Phys-iol. Eehau. 24: 237-242, 1980.
`17. Roy, E. J., and G. N. Wade. Binding of [-’5H]estradioi by brain
`cell nuclei and female rat sexual behavior: inhibition by antiestro
`
`Astrazeneca Ex. 2109 p. 4
`
`

`
`18.
`
`19.
`
`20.
`
`22.
`
`23.
`
`24.
`
`gens. Brain Res. 126: 73-87, 1977.
`Rubin, B. S_., and R. J. Barfield. Priming of estrous respon-
`siveness by implants of 1’Ifii—estradiol in the ventromedial hypo-
`thalamic nucleus of rats. Endocrinology 106: 504~509, 1980.
`Siegel, H. 1-, P. Cohen, and J. S. Rosenblatt. The effect of
`liystenectomy on hormone-induced lordosis behavior in hamsters.
`Physiol. Behuu. 23: 851i8-53, 1979.
`Siegel, H. I.. and J. S. Rosenblatt. Estrogen-induced maternal
`behaviors in ovariectumiaed-hyatarectumized virgin rats. Physiul.
`Behao. 11: 273—2'78, 1978.
`regulation of sociosexual be»
`.Taknhashi, L. K. Hormonal
`havinr in female mammals. Neurasci. Biobehciv. Rev. 14: 403-413,
`1990.
`Wade, G. l\'.. J. D. Blaustein, J. M. Gray, and J. M.
`Meredith. ICI 182,’:'80: a pure antiestrogen that affects behaviors
`and energy balance in rats without acting in the brain. Am. J.
`Physiol 265 (flegulalfory lntegmhue Comp. Phymhl. 34): EH9}!-
`R1398, 1993.
`Wade, G. N., and J. M. Gray. Gonadal effects on food intake
`and adiposity: a metabolic hypothesis. Physiol. Behau. 22: 583-
`593, 1979.
`Wade, G. N., and H. W. Heller. Tamoxifen mimics the effects
`
`ANTIESTROGEN EFFECTS IN HAMSTERS
`
`R1403
`
`26.
`
`27.
`
`of estradiol on food intake, body weight, and body composition in
`rats. Am. J. Phyarlol. 264 (Regulatory Integrative Comp. Phyaiol.
`33): R1219—Rl223, 1993.
`25. Wade, G. N., and J. B. Powers. Tamoxifen antagonizes the
`effects of estradiol on energy balance and estrous behavior in
`Syrian hamsters. Am. J. Pfiysiot. 265 (Regulatory Integrative
`Comp. Physiol. 34); R559—R552, 1993.
`Watie, G. N., and J. E. Schneider. Metabolic fuels and repm~
`duction in female mamma1s.Neuro.5*ci. Biobehav. Rev. 16: 235 272,
`1992.
`Wade, G. N., and I. Zucker. Modulation of food intake and
`locomotor activity in female rats by diencephalic hormone im-
`plants. J. Cump. Physfal. PS3/chul.
`'72: 328-336, 1970.
`Wakeling, A. E., and J. Bowler. ICI-182,780, a new antima-
`trogen with clinical potential. J. Steroid Biachem. Mol. Biol. 43:
`173-177, 1992.
`Wakeling, A. E., M. Dukes, and J. Bowler. A potent specific
`pure antiestrogen with clinical potential. Cancer Res. 51: 3867-
`3873, 1991.
`. Zigmond, R. E.., and B. S. McEwen. Selective retention of
`oestradiol by ceil nuclei in specific brain regions of the ovariecto—
`mized rat. J. Neurochem. 17: 8B9—899, 1970.
`
`28.
`
`'29.
`
`
`
`Astrazeneca Ex. 2109 p. 5