`
`ELSEVTER
`
`Animal Reproduction Science 51 (1998) 81796
`
`ANIMAL
`REPRODUCTION
`SCIENCE
`
`Oestrogenic effects of ICI 182,780, a putative
`anti-oestrogen, on the secretion of oxytocin and
`prostaglandin F20, during oestrous cycle in the
`intact ewe
`
`H.Y. Al-Matubsi a’*, R.J. Fairclough b, G. Jenkin c
`a Centre for Bioproouu ng and Food Technology, Victoria University of Technology, Werribee Campus
`PO Box 14428 MCMC, Melbourne VIC 8001, Australia
`b Department of Biomedical Sciences Victoria University of Technology, 3. Albans Campus PO Box 14428
`MCMC, Melbourne VIC 8001, Australia
`C Department of Physiology, Monash University, Clayton, VIC 3168, Australia
`Accepted 2 February 1998
`
`Abstract
`
`The effect of ICI 182,780, oestrogen antagonist, on the concentrations of oxytocin and uterine
`PGFZa was investigated in intact Border Leicester Merino cross ewes during the late oestrous
`cycle. Twelve cyclic ewes (n = 6 per group) were randomly assigned to receive, at 6 h intervals,
`intra—muscular injection of either peanut oil or ICI 182,780 (1.5 mg kg’1 day’ 1) in oil for 2 days,
`starting at 1900 h on day 13 until 1300 h on day 15 post-oestrus. Hourly blood samples were
`collected via a jugular catheter from 0800 h on day 14 for 37 h and then daily over days 16, 17
`and 18 post-oestrus. Peripheral plasma concentrations of oxytocin, the metabolite of prostaglandin
`FM, 15-keto-13,14—dihydro-prostaglandin FM, (PGFM) and progesterone were measured by
`radioimmunoassay. All ewes treated with ICI 182,780 exhibited functional luteal regression as
`indicated by a marked reduction in plasma progesterone concentrations to less than 1000 pg /ml
`over the period of 18—36 h during sampling period on days 14 and 15 of the oestrous cycle. In
`five of six vehicle-treated ewes, progesterone concentrations declined between day 16 and day 18
`post-oestrus. In the remaining control ewe, progesterone concentrations reach less than 1000
`pg /ml within 36 h of the commencement of the sampling period. During the frequent sampling
`period, the number of oxytocin pulses in the ICI 182,780 treated ewes was significantly higher
`compared to control ewes (2.7 i0.3 vs. 0.8 i0.3). The mean amplitude of oxytocin pulses
`observed was also greater (70.4 i 19.5 pg /ml) in ewes treated with ICI 182,780, but was not
`
`*
`
`author. Tel.:
`Corresponding
`$94103l2@cougar.vut.edu.au
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`+61
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`fax:
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`e-mail:
`
`0378—4320/98/$l9.00 © 1998 Elsevier Science B.V. All rights reserved.
`PII S0378-4320(98)00068-2
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`82
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`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
`
`significantly different from control ewes (33.5 i 12.9 pg /ml). Oxytocin pulses may however have
`occurred following the initial two ICI 182,780 injections but before commencing blood sampling.
`The oxytocin pulses were detected at a mean of 3.2 i 0.2 h following each injection with ICI
`182,780 during blood sampling. In the ICI 182,780-treated ewes, the pulsatile pattern of plasma
`PGFM in jugular blood samples over the 37 h sampling period on days 14 and 15 post-oestrus had
`a higher amplitude (512.9 i 158.9 vs. 121.7 i 78.7 pg/ml) and pulse area (618.1 i 183.3 vs.
`151.5 i 102.9 (pg/ml)7) compared to the vehicle-treated ewes (P < 0.05) respectively. The
`average number of PGFM pulses observed per ewe was 3.0 i 0.7 in the ICI 182,780-treated group
`and was significantly (P < 0.02) higher than the number of pulses (0.5 i 0.3) observed in ewes
`treated with vehicle alone. The PGFM pulses were detected at 4.2 i 0.6 h following each injection
`with ICI 182,780 during blood sampling. The percentage of PGFM pulses that occurred coinci-
`dently with a significant elevation of oxytocin concentrations was 44.4% in ICI 182,780-treated
`compared to 66.6% in control ewes. We conclude that administration of oestrogen antagonist ICI
`182,780 accelerated development of the luteolytic mechanism by enhancing pulsatile secretion of
`oxytocin and PGFM which suggests that ICI 182,780 acts as an agonist for oxytocin and
`prostaglandin FM release in intact ewes when administered at 1.5 mg /kg /day over Day 13 to 15
`post-oestrus. © 1998 Elsevier Science B.V.
`
`Keywords Oestrogen antagonist; Oxytocin; Prostaglandin; Sheep endocrinology
`
`1. Introduction
`
`Considerable evidence now exists indicating that oxytocin can induce endometrial
`prostaglandin F20, (PG)F2a secretion from the uterus (Shanna and Fitzpatrick, 1974;
`Roberts et 31., 1976; Flint et 31., 1990) and that PGFM can stimulate lute31 oxytocin
`secretion (Flint and Sheldrick, 1982; Watkins and Moore, 1987; Lamsa et 31., 1989).
`Pulsatile release of PGFM by the endometrium may be more effective than continuous
`secretion since Schramm et 31. (1983) have demonstrated that exogenous administration
`of PGFM is more potent as 3 luteolysin when administered in a series of pulses rather
`than as a continuous infusion. During luteolysis, oxytocin pulses, or its associated
`neurophysin, occur simultaneously with pulses of PGFM (Fairclough et al., 1980; Webb
`et 31., 1981; Flint and Sheldrick, 1983; Moore et al., 1986; Hooper et 31., 1986).
`Oestrogen also appears to play a role in regulation of uterine function since administra-
`tion of relatively high doses of oestradiol to ewes during the mid-luteal phase (Ford et
`31., 1975; Hixon and Flint, 1987) or to ewes and cows on day 12 (Spencer et 31., 1995;
`Brunner et 31., 1969) resulted in premature lute31 regression and caused a premature
`decline in lute31 weight (Hawk and Bolt, 1970). Oestrogen also appears to affect the
`timing, magnitude and pattern of PGFM response to oxytocin (McCracken, 1980; Zhang
`et 31., 1991; Beard and Lamming, 1994). The mechanism by which oestrogen interacts
`with oxytocin and PGFM at luteolysis is not clear, although time course studies indicate
`that it takes several hours to act.
`
`In a preliminary report, Fairclough et 31. (1988) administered oestradiol 17-13 to
`intact ewes on days 12, 13 and 14 of the oestrous cycle and found an associated increase
`in plasma oxytocin—neurophysin and/ or PGFM with a maximum concentration occur-
`ring at 4—8 h after oestrogen treatment. Furthermore, Mann and Lamming (1995) have
`reported that in ovariectomized cows there was a reduction in the strength of the
`
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`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
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`83
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`luteolytic signal over the later stages of the luteal phase following removal of an
`oestradiol implant on day 11 of the simulated cycle. In heifers, Jacobs et al. (1988) have
`also shown that the increase in secretion of endogenous PGFM that follows induction of
`luteolysis with the PGFM agonist cloprostenol is suppressed by intravenous injections of
`tamoxifen, an oestrogen antagonist. These data indicated that the PGFM secretion at the
`time of luteolysis is dependent on oestradiol.
`Oestradiol-17B is also involved in regulation of the corpus luteum and influences
`luteolysis.
`In ewes, oestrogen may act directly on the corpus luteum through an
`intra-ovarian mechanism because injection of oestrogen directly into the corpus luteum
`at day 10 post-oestrus resulted in regression of the injected corpus luteum but had little
`effect on the contralateral corpus luteum (Cook et al., 1974). Furthermore, removal of
`the main source of oestrogen by destmction of ovarian follicles extended the cycle
`length in the ewe (Karsch et al., 1970; Ginther, 1971; Zhang et al., 1991) and cattle
`(Fogwell et al., 1985; Villa-Godoy et al., 1985). The mechamsm by which oestrogen
`acts to stimulate oxytocin and uterine PGFM is uncertain.
`ICI 182,780 is thought to be a pure steroidal oestrogen antagomst which blocks
`oestrogen action by competing with endogenous oestrogen for oestrogen receptors
`present in the nuclei of oestrogen responsive tissue (Baker and Jaffe, 1996). ICI 182,7 80
`has been found to be devoid of agonist activity in vivo in the immature and mature rat
`(Wakeling et al., 1991) and blocked the production of the 250 kDa protein which was
`associated with the stimulatory action of oestrogen in ovariectomized rat (Yu et al.,
`1996). Recently, Dibbs et al. (1995) have shown that administration of ICI 182,780 to
`ovariectomized rat subcutaneously (1.5 mg/kg daily for 3 days) blocked the effect of
`oestrogen on the uterine oxytocin receptor within 24 h after the final dose. However,
`Wakeling (1993) reported that ICI 182,780 showed no effect on bone density analysis in
`adult female rats, whereas ovariectomy significantly reduced bone density. In guinea-pigs
`the admimstration of 4 mg of the same putative oestrogen antagomst (equivalent to
`4.7—6.15 mg/kg), once a day on days 11—14 of the cycle, significantly reduced the
`output of PGFM from superfused uterine horn in vitro on day 15 post-oestms (Poyser,
`1993). In this case, the ICI 182,780 compound may be acting by increasing receptor
`turnover and reducing the cellular content of oestrogen receptors (Parker, 1993).
`Administration of ICI 182,780 (12 mg) daily for 7 days in women with normal
`menstrual cycles, decrease the incidence of the mid-cycle LH surge and had an
`anti-proliferative effect on endometrial thickness as measured by ultrasound (Thomas et
`al., 1994).
`This study was undertaken to investigate the inhibitory action of the purported
`oestrogen receptor antagomst (ICI 182,7 80) on oxytocin, uterine PGFM production, and
`on luteal function in intact ewes.
`
`2. Materials and methods
`
`2.1. Animals
`
`All protocols were approved by the Animal Experimentation Ethics Committees of
`Victoria University of Technology (AEEC 95/025) and Monash University.
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`84
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`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
`
`Twelve 3-year-old Border Leicester Merino cross ewes, weighing 42—55 kg, were
`used in this study during the breeding season. The oestrous cycles of the ewes were
`synchronised by the insertion of an intra-vaginal controlled internal drug release (CIDR)
`device impregnated with 300 mg progesterone (EAZI-breed CIDR G, Riverina Artificial
`Breeders, Albury, Australia) for 14 days and by an intra-muscular injection of Semm
`Gonadotrophin (400 IU) to each ewe following removal of the CIDR. Ewes were housed
`in an open barn together with a crayon-bearing vasectomized rams. Ewes were checked
`twice daily for behavioural oestms. The day that the ewe display oestrus behaviour was
`designated to be Day 0. One week before experimentation, the ewes were maintained in
`individual pens with access to water and fed luceme chaff, oats and ewe and lamb
`pellets ad libitum.
`On day 13 post-oestrus, cannulation was performed under local anaesthesia (10%
`Lignocaine Hydrochloride Spray: Xylocaine). The jugular vein of each ewe was
`cannulated by insertion of an intracath (Intervenous Catheter Placement Unit; 16 GA;
`Desert Medical, Becton Dickinson, Sandy, UT). The exteriorised end of the catheter was
`connected to a 3 way stopcock. The catheters were secured to the ewes by placing
`Setonet (Elastic net bandage, size 5; Seton Products, England) around the neck over the
`cannula. Cannulae were then maintained for the duration of the experiment by filling
`with heparimsed saline (50 IU/ n11) and used for the collection of blood samples.
`
`2.2. Experimental protocol
`
`Ewes were randomly assigned to two groups (n = 6 per group). One group was
`treated with intra-muscular injections of 1.5 mg kg’1 day’1 ICI 182,780 [7a-[9-
`(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]estra-1,3,5(10)-triene-3,17B-diol]
`(a gift
`from ICI Pharmaceuticals, Australia) in peanut oil while the other group was treated
`with peanut oil alone (controls), at 6 h intervals at 1900 h on day 13 up until 1300 h on
`day 15 post-oestrus. The ICI 182,780 (8.5 mg in 1 ml) was dissolved in absolute ethanol
`to a concentration of 5% (v/v) of the final volume. The exact volume of peanut oil was
`then added and the ethanol was evaporated using mild heat (40°C) and under constant
`stirring with a magnetic stirrer.
`
`2.3. Blood sanpling
`
`Blood samples (5 ml) were collected over days 14 and 15 of the oestrous cycle at
`hourly intervals starting at 0800 h (12 h after commencement of injection of antagomst)
`for 37 h and then daily over days 16, 17 and 18 post-oestrus. Blood samples were
`
`Fig. 1. Effect of ICI 182,780 on oxytocin (. ), PGFM (O), and progesterone (A) concentrations in peripheral
`plasma in intact ewes late in the oestrous cycle (n= 6; ewe 212, 217, 222, 213, 207, 210). ICI 182,780 was
`injected at 1900 h on Day 13 post-oestrus. ICI 182,780 (1.5 mg kg’1 day’ 1) was administered intra-muscu—
`larly every 6 h up until 1300 h on Day 15 post-oestrus. (1) represent time of injection. Zero time (0) h
`represents 0800 h on Day 14. Blood samples were collected hourly starting at (0) h time up to 37 h during
`Days 14 and 15 post-oestrus and, thereafter, once a day on Days 16, 17 and 18 post-oestrus. Statistically
`significant pulses in the oxytocin and PGFM concentrations are identified by (*) and (T), respectively;
`inverted triangles (V) identify synchronous pulses of both compounds.
`
`AstraZeneca Exhibit 2165 p. 4
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`H.Y. AI-Matubsi et aI./Anir‘ra| Reproduction Science 51 (1998) 81—96
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`85
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`collected into polypropylene tubes containing aspirin and ethylenediaminetetraacetic
`acid (EDTA), 0.7 and 0.5 mg/ n11 blood respectively. The catheter was refilled with
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`86
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`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
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`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
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`87
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`centrifuged for 1500 X g for 10 min at 4°C and plasma was separated and stored at
`— 20°C until assayed for oxytocin, PGFM and progesterone by radioimmunoassay.
`
`2.4. Hormone assays
`
`2.4.1. PGFM assay
`Plasma PGFM concentrations were measured by radioimmunoassay (RIA) as previ-
`ously described (Burgess et al., 1990). The antiserum was raised in sheep against PGFM
`conjugated to porcine gelatine and was kindly supplied by Dr. R.I. Cox (CSIRO,
`Blacktown, NSW). At a final dilution of 1:50 000, the cross-reactivity of this antisera
`with other related compounds including 13,l4-dihydro-l5-keto-PGF2a, 6,15-diketo-
`13,14,dihydro-PGF1a, 13,14-dihydro-15-keto-PGE2, 15-keto-PGF2a, lS-keto-PGE2 and
`PGA2 was 100, 4.6, 1.9, 0.9, 0.3, and 0.02%, respectively, and was <0.01% with
`PGFM, PGEZ, 6-keto-PGF1a, 6-keto-PGE1, PGEI, PGD2, PGB2 and thromboxane B2.
`The sensitivity of the assay was 39 pg/ml. The intra- and inter-assay coefficients of
`variation were 7.6% and 9.4%, respectively.
`
`2.4.2. Progesterone assay
`For progesterone, 200 ,ul of jugular venous plasma was extracted with 2 ml n-hexane
`(Crown Scientific, Vic., Australia) as described by Rice et al. (1986). The antisemm,
`raised in sheep against progesterone-1 1 a-Bovine semm albumin (B SA), was generously
`provided by Dr J. Malecki (Regional Veterinary Institute, Department of Agricultural
`and Rural Affairs, Baimsdale, Victoria). At a final dilution of 1:9000, the cross-reaction
`of the antiserum with progesterone, 11a-hydroxy-progesterone, 5a-pregnane-3 a-ol-20-
`one, 5 B-pregnane-3 a-ol-20-one and corticosterone was 100, 43.8, 15.9, 10.0, and
`1.05%, respectively. The cross reactivity was < 1.0% with 11-deoxycortisol, 5a-preg-
`nane-3 a,17a-diol-20-one,
`5 B-pregnane-3 a,17a,
`20 a-triol-20-one,
`5 B-pregnane-
`3a,17a,20 a-triol and 5a-pregnane-3B-ol-2-one, 0.7% with 17a-hydroxy-progesterone,
`< 0.4% with dehydroepiandrosterone, 0.3% with 20 a-hydroxy-pregnene-3-one and
`< 0.2% with cortisol. The sensitivity of the assay was 0.3 ng/ n11. All samples were
`measured in one assay and the intra—assay coefficient of variation was 10.9%.
`
`2.4.3. Oxytocin assay
`For oxytocin, 1.2 ml of the standards or jugular plasma samples were mixed with 5
`ml of chilled (— 20°C) absolute ethanol using a modification of the method described in
`the Australian Laboratory Services radioimmunoassay kit. After centrifugation at 1900
`X g for 20 min at 4°C (Sorvall RT7; AMRAD Pharmacia Biotech, Victoria, Australia),
`the supernatant was decanted and evaporated to dryness with filtered air at 37°C using a
`sample concentrator (Medos, Victoria, Australia). Recovery of 125 I-oxytocin from
`plasma samples after extraction was 80.1 i 1.6% (mean i SEM). The residues of these
`solutions were reconstituted in 1.2 ml of 0.05 M Phosphate buffer (pH = 7.4). Standards
`and samples were incubated for at least 30 min at 4°C. To duplicate aliquots of 500 ,ul
`of the extracted standards and samples, 100 ,ul of 125I tracer (10000 cpm, Du Pont
`Victoria, Australia) and then 100 ,ul of antisera diluted in assay buffer were added. The
`assay was incubated overnight at 4°C. Separation of the bound oxytocin was achieved by
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`H.Y. AI-Matubsi et a|./Anin'a| Reproduction Science 51 (1998) 81—96
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`the addition of 0.05 ml (1 mg) bovine immunoglobulin semm (Calbiochem, Victoria,
`Australia) and 1 ml of 15% polyethylene glycol 6000 (Crown Scientific, Victoria,
`Australia) at 4°C and then spun at 3300 X 9 rpm for 20 min. The supernatant was
`aspirated and the radioactivity present in the precipitate was quantified using a Packard
`Crystal Gamma Counter (Wallac, Wizard 1470, The Australian Chromatography, Victo-
`ria, Australia). The antisemm to oxytocin (GJ, 137/ 1) was raised in sheep and was
`kindly donated by Dr A.P.F. Flint (Umversity of Nottingham, UK) and was used at a
`final dilution of l:35,500. The cross-reaction of this antiserum with oxytocin, isotocin
`and melanocye-stimulating hormone (MSH) was 100, 0.8, and 0.04%, respectively. The
`cross reactivity was < 0.02% with mesotocin, pressinoic acid, prolactin, argimne
`vasopressin and lysine vasopressin. The sensitivity of the assay was 10 pg/ n11, the intra-
`and inter-assay coefficients of variation were 11.7% and 13.2%, respectively.
`
`2.5. Satistioal analysis
`
`Statistically significant pulses of peripheral oxytocin and PGFM concentrations were
`identified using a Pulsar program (Merriam and Wachter, 1982). Assay noise was
`estimated by regression analysis of the standard deviation for the duplicate determina-
`tions and the mean at each point. Base line was calculated as representing the
`contribution of circadian rhythms or other long term trends but not fluctuations of
`shorter duration. The amplitude of the peripheral oxytocin and PGFM pulses were
`calculated by subtracting base-line values and then rescaled in terms of standard
`deviation umts by dividing the rescaled values by an estimate of assay noise. The
`amplitude of the rescaled pulses were then identified by applying height and duration
`criteria specified by user-defined cut-off points [G( n)] for pulses. These calculations
`were repeated until 2 iterations produced the same values for pulses or until the preset
`limit, 6 iterations, was completed. The quadratic (a), linear (b), and constant (C) terms
`for pulsar were as follows: for oxytocin, a = 0.00, b = 13.15, C = 0.00; and for PGFM,
`a = 0.00, b = 9.44, C: 0.00. The following G( n) values were selected for oxytocin
`pulses: G(1)= 3.8, G(2) = 2.6, G(3) = G(4) = G(5) = 1.2; and for PGFM pulses:
`G(l) = 4.5, G(2) = 2.6, G(3) = 1.9, (4) = 1.5, G(5) = 1.2. The oxytocin and PGFM
`pulses were identified as significant pulses if they were higher than 50 pg/ n11 and 200
`pg/ml, respectively. Coincident episodes in the secretion of oxytocin and PGFM were
`defined as those which showed an increase in the value of the PGFM pulse with a
`defined oxytocin pulse. The area under the significant peripheral oxytocin and PGFM
`pulses were calculated for each ewe and was expressed for both hormones as (pg/ml)7'.
`
`Fig. 2. Effect of vehicle injection on oxytocin (. ), PGFM (O), and progesterone (A) concentrations in
`peripheral plasma in intact ewes late in the oestrous cycle (n = 6; ewe 211, 218, 219, 208, 205, 216). Vehicle
`was injected at 1900 h on Day 13 post-oestrus. Vehicle was administered intra-muscularly every 6 h up until
`1300 h on Day 15 post-oestrus. (1) represent time of injection. Zero time (0) h represents 0800 h on Day 14.
`Blood samples were collected hourly starting at (0) h time up to 37 h during Days 14 and 15 post-oestrus and,
`thereafter, once a day on Days 16, 17 and 18 post-oestrus. Statistically significant pulses in the oxytocin and
`PGFM concentrations are identified by (*) and ( T), respectively; inverted triangles (V) identify synchronous
`pulses of both compounds.
`
`AstraZeneca Exhibit 2165 p. 8
`
`

`

`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
`
`89
`
`
`300
`
`1
`
`1
`
`i
`
`1
`
`L
`
`*
`
`10000
`9000 —
`8000 ~
`
`7000 _
`6000 —
`5000 —
`
`4000 —
`3000 —
`2000 —
`1000 —
`0
`
`250 —
`
`200 _
`
`150 —
`
`100 _
`
`so —
`
`0
`
`211— 2200
`— 2000
`— 1800
`
`— 1400
`— 1600
`— 1200
`— 1000
`
`_ 000
`
`—
`A
`E
`Ea
`g
`E
`(5
`n.
`
`219— 2200
`~ 2000
`
`— 1800
`
`— 1400
`- 160°
`— 1200
`— 1000
`
`_ 800
`
`
`
`
`
`
`
`0
`
`
`
`10000 —
`9000 —
`
`300 —
`
`25° _
`
`A
`8000 —
`— —
`_ m
`7000 E 200 _
`6000 g
`5000 E 150
`°
`4000 — 5;
`3000 — X
`O
`2
`0 —
`00
`1000 -
`0
`
`100 _
`
`50 _‘
`
`0
`
`
`
`Progesterone(pg/ml)
`
`
`300
`
`10000
`9000 —
`
`250 —
`
`200 _
`
`150 —
`
`100 _
`
`8000 _4
`
`7000 —
`6000 ——
`5000 —
`
`4000 —
`3000 —
`2000 —
`1000 A
`
`14
`
`I
`
`15
`
`|16|17 I18|
`
`Days post-oestrus
`
`AstraZeneca Exhibit 2165 p. 9
`
`

`

`8000 —
`25° -
`”E
`7000 _ E
`B)
`ca
`\ 20° —
`g
`6000 — Q
`0:
`V
`c:
`5000
`.E
`o
`8
`E
`E 4000— 1;
`c»
`3000 — X
`9
`O
`o_
`
`
`
`
`100—
`
`50 *
`
`— 1500
`— 1000
`— 1400
`— 1200
`
`PGFM(pg/m1)
`
`
`
`90
`
`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
`
`(D) 10000 —
`9000 —
`
`300 ——
`
`i
`
`I
`
`i
`
`i
`
`1
`
`i
`’
`
`3000 _
`
`7000 —
`6000 -
`5000
`
`4000 —
`3000 —
`2000 —
`1000 —
`
`250 _
`
`200 _
`
`150
`
`10° _
`50 —
`
`0
`
`0
`
`l
`
`'
`
`208— 2200
`— 2000
`
`— 1800
`
`1600
`14001200
`— 1000
`
`800
`
`10000 —
`9000 ——
`
`300 —
`
`T
`
`V
`
`205— 2200
`— 2000
`
`A
`
`150
`
`
`
`
`
`
`— 1000
`_800
`— 600
`2000 -
`_ 400
`1000 —
`— 200
`
`
`0
`I
`I
`I w 0
`
`0
`
`10000 —-
`9000 —
`
`300 —
`
`216— 2200
`— 2000
`
`8000 —
`
`7000 —
`6000 _
`5000 —
`
`4000 —
`3000 —
`2000 —
`1000 —
`
`25° _
`
`200 _
`
`150 —
`
`10° _
`50 _
`
`
`
`
`°
`
`0
`
`I—'—‘—I—'—l
`
`I
`
`
`
`
`— 1800
`
`‘ 1600
`— 1400
`— 1200
`— 1000
`
`800
`_ 600
`— 400
`— 200
`' ‘.~.~.~.~.r.~. egg
`
`I
`I
`I
`I
`I
`I
`I
`6
`12
`18
`24
`30
`36
`(hrs)
`
`0
`
`14
`
`I
`
`15
`
`I16 l17l1al
`
`Days post-oestrus
`
`Fig. 2 (continued).
`
`AstraZeneca Exhibit 2165 p. 10
`
`

`

`H.Y. AI-Matubsi et aI./Anirm| Reproduction Science 51 (1998) 81—96
`
`91
`
`The plasma concentrations of oxytocin and PGFM were determined as pg/ n11 while the
`length of that pulse was designated as 7'. Estimated parameters included overall mean
`concentrations, base value, pulse number, pulse amplitude and area under the pulse and
`were carried out using the Pulsar analysis program. The mean i SEM of these parame-
`ters were calculated from all ewes showing significant pulses of either oxytocin and/or
`PGFM and were compared using a Student’s unpaired t-test.
`
`3. Results
`
`Individual patterns of progesterone secretion during the 37 h sampling period on days
`14 and 15; and on days 16, 17 and 18 of oestrus are shown in treated and control ewes
`in Figs. 1 and 2, respectively. Plasma concentrations of progesterone at the beginning of
`the sampling period in ICI 182,780 and vehicle-treated ewes ranged from 2064.7 to
`9046.6 pg/ml, indicating that a functional corpus luteum was present in each ewe at the
`start of experiment.
`The time required for progesterone to reach concentrations of less than 1000 pg/ n11
`in ICI 182,780-treated ewes was significantly (P < 0.005) shorter than that for control
`ewes. All ewes treated with ICI 182,7 80 showed functional luteal regression as indicated
`by progesterone concentrations (< 1000 pg/ n11) in peripheral plasma which declined
`sharply between 18—36 h during the sampling period on days 14 and 15 of the oestrous
`cycle. In five of the six vehicle-treated ewes, progesterone concentrations started to
`decrease on or after day 16 post-oestrus. In one of the control ewes, progesterone
`concentrations reached less than 1000 pg/ n11 at 36 h after the start of the sampling
`period (Fig. 2).
`Mean basal peripheral plasma concentrations of oxytocin in ICI 182,7 80-treated ewes
`were 22.3 i 7.7 pg/ml and not significantly (P > 005) different than those measured in
`animals treated with vehicle alone (23.8 i 7.9 pg/n11) over sampling period. Mean basal
`peripheral plasma concentrations of PGFM were also not significantly (P > 0.05)
`different in ICI 182,780-treated ewes (40.3 i 1.0 pg/ n11) compared with those ewes
`which received vehicle alone (39.4 i 0.4 pg/ ml) over sampling period.
`The effect of intra-muscular injections of ICI 182,780 and vehicle on peripheral
`oxytocin and PGFM concentrations are shown in Figs. 1 and 2, respectively. The results
`of this study clearly demonstrate that administration of ICI 182,7 80 to intact ewes every
`6 h for 2 days significantly (P < 0.01) increased the number of oxytocin pulses per ewe
`(2.7 i 0.3) compared to control ewes (0.8 i 0.3). However, the mean amplitude (70.4 i
`19.5 pg/ml) and the mean area (95.9 i 23.9 pg/ml)7' of oxytocin pulses in ewes
`treated with ICI 182,780 were not significantly different compared to corresponding
`values of 33.5 i 12.9 pg/ml and 79.8 i 34.4 (pg/ml)7, respectively. In ICI 182,780-
`treated ewes, at least two detectable pulses in plasma oxytocin concentration were
`observed per ewe during the sampling period with a maximum concentration occurred at
`the start of the sampling period and then gradually declined during luteolysis. The
`oxytocin pulses were detected at a mean of 3.2 i 0.2 h following each injection with ICI
`182,780. In two of the six control ewes, no detectable pulse of oxytocin was found
`during the sampling period on days 14 and 15 of the oestrous cycle. In the remaining
`
`AstraZeneca Exhibit 2165 p. 11
`
`

`

`92
`
`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
`
`four ewes, one pulse of oxytocin was observed per ewe over days 14 and 15 post-oestrus
`apart from one ewe (218; Fig. 2) which showed two pulses during the same period. In
`these ewes, the oxytocin pulses were detected at a mean of 5.3 i 0.5 h following each
`injection with vehicle during blood sampling.
`The pulsatile pattern of plasma PGFM in jugular blood samples over the 37 h
`sampling window on days 14 and 15 post-oestms is shown in (Figs.
`1 and 2). The
`average number of PGFM pulses observed per ewe in ICI 182,780-treated group was
`3.0 i 0.7 and significantly (P < 0.02) higher than those observed in ewes treated with
`vehicle alone (0.5 i 0.3). There was a significant differences (P s 0.05) in amplitude
`(512.9 i 158.9 vs. 121.7 i 78.7 pg/ml) and pulse area (618.1 i 183.3 vs. 151.5 i 102.9
`(pg/ml)7-) of the PGFM response measured in ICI 182,780-treated ewes compared to
`that in vehicle-treated ewes, respectively.
`Five of SIX ewes given ICI 182,780 showed at least two detectable pulses in plasma
`PGFM concentration during sampling period. In the remaining ewe (217; Fig. 1) one
`detectable pulse of PGFM was observed in the peripheral plasma. The first pulses of
`PGFM in the ICI 182,780 group were relatively low in magmtude and then increased
`during the sampling period. In these ewes the PGFM pulses were detected at a mean of
`4.2 i 0.6 h following each injection with ICI 182,780 during blood sampling. In four of
`SIX control ewes, no significant pulses in plasma PGFM concentrations were found
`during the sampling period. In one of the other two control ewes, one pulse of plasma
`PGFM was observed (218; Fig. 2) and in the other ewe (205; Fig. 2) there were two
`pulses of PGFM in peripheral plasma. The PGFM pulses were detected in these two
`ewes at a mean of 5.3 i 0.8 h following each injection with vehicle during blood
`sampling. The percentage of PGFM pulses that occurred coincidently with a significant
`elevation of oxytocin concentrations was 44.4% in ICI 182,780-treated, compared to
`66.7% in control ewes, although this difference was not statistically significant (P >
`0.05).
`
`4. Discussion
`
`This study is the first to describe the effect of ICI 182,780 administration on both
`oxytocin and PGFM release in intact ewes. A major finding of this study was that
`premature functional luteolysis occurred in all ewes treated with ICI 182,780 during
`days 14 and 15 of oestrous cycle, as indicated by a fall in progesterone levels; whereas,
`in five out of SIX vehicle-treated ewes, functional luteolysis was not observed until
`between days 16 and 18 post-oestms.
`Previous studies (McCracken, 1980; Hixon and Flint, 1987; Beard and Lamming,
`1994; Al-Matubsi et al., 1998) indicate that oestrogen affects the timing, magmtude and
`pattern of PGFM response to oxytocin. Mann and Lamming (1995) have demonstrated
`previously that a low level of oestradiol admimstered over the second half of the luteal
`phase in the ovariectomized cow model leads to a weak luteolytic signal. However,
`Zhang et al. (1991) found that there was a tendency for the amplitude of PGFM pulses
`to be lower in oestradiol-treated than in control ewes without any effect on the number
`of PGFM pulses.
`
`AstraZeneca Exhibit 2165 p. 12
`
`

`

`H.Y. AI-Matubsi et al ./Animal Reproduction Science 51 (1998) 81—96
`
`93
`
`If oestrogen is associated with the luteal increase in oxytocin and PGFM pulses, the
`finding that the average number of plasma PGFM pulses observed per ICI 182,7 80-treated
`ewe was significantly (P < 0.02) higher than those observed in ewes which received
`vehicle alone between days 14 and 15 post-oestrus, indicates that ICI 182,780 failed to
`antagonise oestradiol secretion in intact ewes. This increase in the number of PGFM
`pulses may have been the cause of premature luteolysis, since it has been shown that the
`frequency at which PGFM is secreted is a factor in determining its effectiveness in
`causing luteolysis (Zarco et al., 1984; Zhang et al., 1991).
`The percentage of PGFM pulses that occurred coincidently with a significant
`elevation of oxytocin concentrations was decreased by the admimstration of ICI
`182,7 80. This view gives further support to the view of Zhang et al. (1991) who reported
`similar effect of oestradiol admimstered to ewes treated with either sham or X-irradiated
`
`ovarian follicles. Thus, these observations indicate that ICI 182,780 may be acting to
`mimic the action of oestrogen in stimulating oxytocin and prostaglandin F201 release in
`intact ewes.
`
`the concentrations of
`Flint and Sheldrick (1983) have previously reported that
`oxytocin following synthetic prostaglandin treatment were higher in ovarian (219— 19230
`pg/ml) than in jugular (23.9—183.1 pg/ n11) plasma and that release was absent
`following ovariectomy. The amplitude of the jugular oxytocin pulses (28.3—281.6
`pg/ml) in the present study is comparable to that observed by Flint and Sheldrick
`(1983) and to those detected by Hooper et al. (1986) in jugular vein (20—220 pg/ n11) in
`which they employed sampling intervals of 1 h for 12 consecutive hours over days
`13—16 of oestrous cycle in cycling ewes. These observations indicate that the concentra-
`tion of oxytocin detected in this study represents luteal rather than posterior secretion.
`If this compound is acting as an oestrogen agonist in the ewe, the data obtained in
`this study of an increase in the frequency of oxytocin pulses from peripheral plasma in
`ewes give