Biological Activity and Steroid Receptor Interactions of Pituitary Gland And

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Biological activity and steroid receptor interactions of cyclofenil with the oestrogen target tissues of the brain, pituitary gland and uterus of the rat

S. P. Bowman, A. Leaket and I. D. Morris

Department ofPharmacology, Materia Medica & Therapeutics, Stopjord Building, Manchester University, Oxford Road, Manchester Ml3 9PT, U.K.

Summary. In vitro cyclofenil [bis-(p-acetoxyphenyl)-cyclo-hexylidene methane] competitively inhibited the binding of [3H]oestradiol-17\g=b\to the uterine cytoplasmic receptor (Kdi = 9\m=.\41nmol/l). By 24 h after cyclofenil treatment of ovariectomized rats, cytoplasmic oestrogen receptors were altered, in a similar manner, in brain, pituitary gland and uterus. The lowest dose depleted the receptor concentration which was re-established at the intermediate dose and depleted again after the highest dose. Nuclear oestrogen receptors measured in the uterus were raised only after the highest dose of cyclofenil. Uterine cytoplasmic progesterone receptors were increased after cyclofenil, the concentration of progesterone receptor after the high dose of cyclofenil being greater than after a large dose of oestradiol-17\g=b\or oestradiol-17\g=b\benzoate. Cyclofenil was uterotrophic; the increase in uterine weight was apparently dose-related and at 24 and 48 h the increase was greater or similar to the increases produced by oestradiol-17\g=b\or oestradiol-17\g=b\benzoate. Accumulation of uterine luminal fluid was also observed but oestradiol-17\g=b\benzoate was more active in this respect. Cyclofenil antagonized all the uterine effects of oestradiol-17\g=b\ benzoate in the immature female rat but only the changes in uterine luminal fluid in the adult ovariectomized rat. Cyclofenil decreased serum LH and increased serum FSH concentrations, effects similar to those of oestrogen except when administered as three daily doses when, unlike oestrogen, no changes in FSH were observed. Antagonism of the activity of oestrogen upon the serum gonadotrophins was not seen. Serum prolactin concentrations were increased 24 h after 0\m=.\5and 50 mg cyclofenil/kg but not after 10 mg/kg. Cyclofenil was not active in inducing sexual receptivity in ovariectomized adult rats unless previously treated with progesterone when a high level of activity was observed. The results show that the cyclofenil molecule possesses high oestrogenicity which is probably mediated via the oestrogen receptor system. This would suggest that the mechanism by which cyclofenil induces ovulation is most probably related to its oestrogenicity rather than its antioestrogenicity.

* Present address: Clinical Research Laboratory, Christie Hospital & Holt Radium Institute, Wilmslow Road, Manchester M20 9BX, U.K. t Present address: Department of Surgery, Medical School, University of Edinburgh, Teviot Place, Edinburgh EH8 9AG, U.K.

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access Introduction Cyclofenil [bis-(/?-acetoxyphenyl)-cyclo-hexlidine methane] has been extensively employed to induce ovulation in women in whom there is a functional disturbance of the hypothalamic- pituitary axis (Berger, 1972; Elmendorff & Kammerling, 1977). Ovulation may also be induced by clomiphene and tamoxifen and the pregnancy rates that have resulted from the three drugs are similar, which suggests that they have a common mode of action (MacGregor, Johnson & Bunde, 1968; Elmendorff & Kammerling, 1977). Clomiphene and tamoxifen have been shown to antagonize oestrogen by interacting with the oestrogen receptor system of the target cells. The oestrogen antagonists combine with the cytoplasmic receptor protein and the complex is trans¬ located to the nucleus; however, the nuclear 'complex' is inactive and unable to induce the oestrogenic response. Part of this response is the resynthesis of cytoplasmic receptor, and the inhibition of this response, combined with the long plasma half lives of these drugs, produces low levels of cytoplasmic receptor and the tissue becomes refractory to oestrogen (Clark & Peck, 1979; Furr, Patterson, Richardson, Slater & Wakeling, 1979; Katzenellenbogen et al, 1980). Cyclofenil is often grouped with the triarylethylene derivatives such as clomiphene and tamoxifen as an oestrogen antagonist and the basic mechanism by which these drugs are believed to induce ovulation is through oestrogen antagonism at the receptors of the hypothalamus and pituitary (Bishop, 1970; Lunan & Klopper, 1975; Furr et al. 1979; Leclercq & Heuson, 1979). However, it is possible that the mode of action of cyclofenil differs, because cyclofenil is administered continuously, while a short course of therapy is recommended for clomiphene and tamoxifen. In women the agonistic activity of clomiphene and tamoxifen is rarely seen and clomiphene produces a thick, scanty cervical mucus; in contrast, cyclofenil would appear to act as an agonist upon the cervix, producing mucus that is thin and copious (Sato, Ibuko, Hirondo, Igarashi & Matsumoto, 1969; Murray & Osmond-Clarke, 1971). The biological properties of cyclofenil do not appear to be well defined in the literature. Cyclofenil is both oestrogenic and antioestrogenic. In rodents, cyclofenil is uterotrophic and will inhibit gonadotrophin secretion, as indicated by atrophy of the gonads of either sex (Einer-Jensen, 1968; Carlborg, 1970). Einer-Jensen (1968) reported that cyclofenil would not produce the same maximum uterine weight as oestradiol benzoate, which suggests that the cyclofenil was a partial oestrogen agonist. Antiuterotrophic activity was demonstrated by Watnick & Neri (1968). Devleeschouwer, Leclercq, Danguy & Heuson (1978) described the ability of cyclofenil, as an antioestrogen, to produce mammary tumour regression in the rat, although oestrogens and antioestrogens can cause tumour regression. As part of a study designed to investigate the mechanism by which the oestrogen antagonists induce ovulation, the receptor interactions and the biological activity of tamoxifen, clomiphene and cyclofenil are being compared. Clomiphene and tamoxifen interact with the cytoplasmic oestrogen receptor of the brain and pituitary gland, but in vivo larger doses are required to affect the hypothalamic oestrogen receptors, access of the drug being prevented by the blood-brain barrier (Ginsburg, MacLusky. Morris & Thomas, 1977; Kurl & Morris, 1978). This paper describes experiments which were performed to see whether the oestrogen antagonist cyclofenil could be shown to interact with the oestrogen receptor system of the brain and pituitary, as well as the uterus, and whether exclusion of the drug from the brain is associated with all the ovulation-inducing antioestrogens. In addition, experiments were conducted to help define the biological activity of cyclofenil so that a comparison could be made with the more extensively investigated triarylethylene oestrogen antagonists.

Materials and Methods

Female Sprague-Dawley rats bred in the Medical School were used. Immature rats were 25 days old (approximately 60 g) and were used without further treatment. Adult female rats

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access (approximately 290 g) were bilaterally ovariectomized and used 2 or 3 weeks later. The rats were kept under standard animal house conditions (lights on 05 :00-19 :00 h) and allowed food and water ad libitum. At the appropriate time cyclofenil (0-5-50 mg/kg; Roussel, Wembley Park, U.K. or Ferrosan, Malmo, Sweden), oestradiol-17ß (72 µg/kg; Sigma, Poole, Dorset, U.K.)or oestradiol- 17ß benzoate (100 µg/kg; Sigma) was administered after solution or suspension in warm arachis oil. Control animals received arachis oil only (1 ml/kg s.c). Cyclofenil was administered intra- peritoneally while oestradiol-17ß and oestradiol-17ß benzoate were administered subcutaneously. The dose of oestradiol benzoate was adjusted so that the mass of oestradiol-17ß admin¬ istered to the rats was identical in each oestrogen treatment. Animals were killed by stunning and decapitation. Serum was prepared and then stored at 15°C until required for the assay of gonadotrophins. The uterine horns were ligated at both — ends in situ, dissected out and weighed. The amount of uterine luminal fluid was determined by the difference in weights after cutting the uterine horns, blotting the tissue dry and reweighing. The hypothalamus, pituitary gland and amygdala were dissected out as described previously (Ginsburg, Greenstein, MacLusky, Morris & Thomas, 1974), and weighed, and subcellular fractionation of the 4 tissues was carried out as described below.

Cytoplasmic oestrogen receptor assay Homogenization, cytosol preparation and the determination of the number of oestrogen receptors and their affinity for oestradiol-17 ß was carried out as described by Ginsburg et al. (1974). Briefly, the tissues were homogenized in 0-01 M-phosphate buffer, pH 7-4, containing 0-1 M-2-mercaptoethanol and 0-25 M-sucrose. Homogenates were centrifuged at 105 000 g for 1 h at 4°C. Aliquots of the cytosol fraction were incubated at 30°C to equilibrium with 4 10~9- 2 10~10 M-[3H]oestradiol-17ß (sp. act. 90 Ci/mmol; The Radiochemical Centre, Amersham, Bucks, U.K.) with or without a 100-fold excess of diethylstilboestrol. When appropriate, non-radioactive compounds dissolved in absolute ethanol were added to the incubate (final concentration 3% v/v). After incubation, bound and free oestradiol were separated by the use of small columns of Sephadex LH-20 maintained at 4°C. The radioactivity in the eluates from this and from the cytoplasmic progestagen and nuclear oestrogen assays was determined by liquid scintillation counting. Tissues were also homogenized in 0-1 M-Tris-HCl buffer, pH 7-6 (Tris buffer) containing 0-01 M-EDTA, 0-03 M-MgCl2, 0-5 mM-dithiothreitol, 0-5 mM-bacitracin and 10% glycerol for the determination of cytoplasmic progestagen and nuclear oestrogen receptors. The homogenate was centrifuged at 1000 g for 10 min at 4°C. The supernatant was removed and centrifuged at 105 000 g for 1 h at 4°C to produce the cytosol fraction to be assayed for progestagen receptors. The pellet from the 1000 g centrifugation step was used for the determination of nuclear oestrogen receptors.

Nuclear oestrogen receptor assay The method of Roy & McEwen (1977) for the assay of nuclear oestrogen receptors involved the preparation of purified cell nuclei which were lysed with a hypotonie buffer and then treated with Tris buffer, pH 7-6, containing 0-4 M-KC1, 0-5 mM-dithiothreitol and 0-5 mM-bacitracin (Tris-KCl buffer) to extract the receptor. Aliquots of the extract were then incubated with PHloestradiol (3 10 8-3 "10 m) for 2 h at 30°C with or without a 100-fold excess of diethylstilboestrol. Separation of bound from free oestradiol was carried out using Sephadex LH-20 columns as described above.

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access Progestagen receptor assay

Progestagen receptors in the uterine cytosol were measured by the method of MacLusky & McEwen (1980). Aliquots of the cytosol were incubated with [3H]promegestone (0-6-20 10~9 m) (sp. act. 80 Ci/mmol: N.E.N. Chemicals, Dreiech, West Germany) in Tris-KCl buffer with or without a 100-fold excess of promegestone for 3 h at 4°C. Bound and free promegestone (17a-methyl-17-propionyl-estra-4,9-dien-3-one) were separated on Sephadex LH-20 columns as described above. All non-radioactive chemicals were obtained from Sigma. The assayed concentrations of specifically bound oestradiol-17ß or promegestone in the incubates were used to construct plots (Scatchard, 1949) from which the equilibrium dissociation constant (Kd) and the saturation binding capacity of the cytosol were determined. After the administration of 50 mg cyclofenil/kg, reliable Scatchard plots could not be constructed for [3H]oestradiol-17ß binding because of the low levels of specifically bound oestradiol. Therefore to obtain an estimate of the saturation binding capacity the specifically bound oestradiol was determined for the two highest [3Hloestradiol concentrations and estimated from this value; Kd could not be determined for these cytosols. Binding of the ligands to the cytoplasmic receptors is expressed per mg protein, the latter determined by the method of Lowry, Rosebrough, Farr & Randall (1951). The binding of [3H]oestradiol to the nuclear extract is related to the DNA content of the pellet remaining after extraction of the receptor. DNA was determined fluorometrically by the method of Kapuscinski & Skoczylas (1977).

Hormone assays

Prolactin, LH and FSH were assayed by the double-antibody technique using reagents supplied by the National Pituitary Agency, NIAMDD, U.S.A. Details of the LH and FSH radioimmunoassay are given by Morris (1979); the within-assay coefficients of variation and sensitivities were 7-5 and 5-5% and 7 and 70 ng/ml respectively. The reference preparation and antibody for the prolactin assay were NIH-PRL-RP-2 and S-7 respectively, and the within-assay coefficient of variation and sensitivity were 6% and 0-3 ng/ml respectively.

Sexual receptivity

The effect of the drug treatment upon sexual receptivity was examined in female rats ovariectomized 3 weeks previously and housed in reversed lighting (lights on 20:00-08:00 h). Female rats were tested in the dark phase of their light cycle and were placed in a semicircular arena (diam. 1 m) with 2 vigorous male rats. The rats were observed under red light and the number of mounts achieved by the male and the times lordosis was shown by the female were counted. After 10 mounts the female was removed. The results are expressed as the percentage lordosis quotient, i.e. (no. of times lordosis shown/no. of mounts) 100. Progesterone (4 mg/kg: Sigma) was administered subcutaneously, dissolved in arachis oil, when indicated.

Statistical analysis

The results are expressed as the means ± s.e.m. and the significance of the difference between groups was calculated by the Mann-Whitney U test (two-tailed).

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access Results Steroid receptors In-vitro experiments. The effects of cyclofenil on the in-vitro binding of [3Hloestradiol-17ß to cytosol prepared from the uteri of rats ovariectomized 3 weeks previously was examined. Cyclofenil produced a dose-related inhibition of the [3Hloestradiol (1 10~9 m) binding (Text-fig. la). The affinity of cyclofenil for the cytoplasmic receptor was estimated by construction of binding isotherms for PHloestradiol to the cytosol with and without cyclofenil. When the data were plotted according to the method of Scatchard (1949) (Text-fig. lb),

O [3Hloestradiol-17ß •fa [3H]oestradiol + 6-7 X 1 O"10 M-diethylstilboestrol • (3H]oestradiol + 3-6 10~9 M-cyclofenil

0036 3-60 360 12 3 4 Cyclofenil 10~7 M Bound [3H]oestradiol (Ci 1CT9) Text-fig. 1. The effect (a) and Scatchard analysis (b) of cyclofenil on the binding of [3H]oestradiol-17ß to incubates (0-2 ml, 1 mg protein/ml) of cytosol prepared from the uteri of rats ovariectomized 3 weeks earlier, (a) Values are mean + s.e.m. for = 4. cyclofenil produced a decrease in the slope of the line. However, the intercept on the abscissa was similar to that produced by [3H]oestradiol alone, indicating that PHloestradiol and cyclofenil were interacting with the same binding site. The effect of diethylstilboestrol is also given for comparative purposes. Estimation of the apparent dissociation inhibitory constant (Kd[, Edsall & Wyman, 1958) gave values of 9-41 nmol/1 for cyclofenil, 0-094 nmol/1 for diethylstilboestrol compared with the apparent dissociation constant (Kd) of 0-415 nmol/1 for PHloestradiol. In-vivo experiments. Cyclofenil produced dose-related changes in the cytoplasmic receptor concentrations estimated 24 h after administration of the drug (Table 1). A fall in the receptor concentration was produced by 0-5 mg cyclofenil/kg which was significant in all tissues (P < 0-05) except the uterus. There was a gradual return of receptor concentrations to control values between the doses of 1 and 10 mg/kg. The apparent increases above control values were significant for the uterus at 5 and 10 mg cyclofenil/kg (P < 0-05) and the amygdala at 10 mg/kg (P < 0-05). Tissue receptor concentrations were markedly reduced (P < 0-01) in all tissues 24 h after 50 mg cyclofenil/kg. The Kd for the reaction between PH]oestradiol-17ß and the

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access cytoplasmic receptor prepared from the control animals, expressed in nmol/1 were: amygdala, 0-284 ± 0-054; anterior hypothalamus, 0-295 ± 0-048; pituitary gland, 0-244 + 0-06; and uterus, 0-296 + 0-072 (n = 5-6). The treatment of the rats with cyclofenil before killing did not significantly change the dissociation constants except after 10 mg cyclofenil/kg, for the anterior hypothalamus, which was raised to 0-767 + 0-264 (n = 4, < 0-05), and for the pituitary gland which was raised to 0-684 ± 0-024 (n = 3, < 0-05).

Table 1. Effect of cyclofenil on the concentrations of cytosolic oestrogen receptors in adult ovariectomized rats

Oestrogen receptors (per mg cytosol protein 10"') Cyclofenil Anterior Middle Pituitary (mg kg"1) Amygdala hypothalamus hypothalamus gland Uterus

0 (control) •14 + 0-49(5) 39 ±1-44(6) ·08±2·49(5) 124 3 + 40 (6) 80-6 ±23-5 (6) 0-5 •43+ 0-39* (5) 58 + 0-63**(5) -76±0-49*(5) 46 4+10-7* (5) 43-4±20-4(5) 10 86 + 0-81(3) •85 ±1-57 (4) •39 ± 1-97(4) 116 5 + 40 (5) 144 ±41 (5) 5-0 •34±0-81(4) •44 ±0-94 (5) 86±0-87(5) 140 4 + 33(5) 175 ±20 (5) 10 •31±1-1*(6) •84± 1-32(6) -51 ± 1-2 (6) 170 8 ± 26 (6) 162 ±46* (6) 50 •91 ±0-45** (4) •84 ±0-21** (6) 78±0-36**(6) 26 4 ±13-5** (6) 3-9±l-5**(6)

The rats were killed 24 h after administration of cyclofenil. Control oestrogen receptor concentrations were determined in cytosols prepared from animals given vehicle (arachis oil) only. Values are mean + s.e.m. for the no. of determinations given in parentheses. Values significantly different from the control value, *P < 0-05; **P < 0-01 (Mann-Whitney U test, two-tailed).

Nuclear oestrogen receptors were measured after extraction of the receptors from nuclei prepared from the uteri of cyclofenil-treated rats (Table 2). A significant 4-fold increase in nuclear receptors was detected after 50 mg/kg, the lower doses having no effect. In comparison a 6-fold increase in nuclear receptors was detected 1 h after oestradiol-17ß. Significant changes were not observed for the apparent dissociation constant, but the affinity of oestradiol for the nuclear oestrogen receptor was approximately 10-fold less than that measured in the cytoplasm prepared from uteri of rats which received cyclofenil, oestradiol or oestradiol benzoate (Table 2). Increases in the progesterone receptor concentrations were detected after all the doses of

Table 2. Uterine nuclear oestrogen receptor and cytoplasmic progestagen receptor concentrations and their respective dissociation constants 24 h after cyclofenil treatment of ovariectomized rats Nuclear oestrogen receptors Cytoplasmic progestagen receptors Dissociation Dissociation Sites per constant Sites per constant µ$ DNA 10" (mol/1 10 10) mg protein 10" (mol/1 IO"10) Control 2-78 ±0-75 16-8 ±4-2 18-4 ±5-6 5-21 ±2-40 Cyclofenil (mg/kg) 0-5 2-84 ±0-66 13-4 + 4-4 52-3 ±24-4 4-65 ±0-87 10 4-75 ± 1-40 15-7 ±3-6 108-6 ± 12-7** 9-07 ±2-50 50 12-98 ±3-21" 38-3 ± 11-1 174-0 ± 53-2** 7-42 ± 1-92 Oestradiol (20 µg/kg) 1 h 18-9 ±3-27** 31-6 ± 9-2 16-9 ± 3-2 4-44 ± 2-05 24 h ND ND 80-7+ 3-0" 6-06 ± 2-78 Oestradiol benzoate (100 Mg/kg) 24 h ND ND 37-3 + 12-0 4-67 ±0-8

Values are mean + s.e.m. for 4-7 observations. ND, not determined. Significantly different from control values, **P < 0-01 (Mann-Whitney U test, two-tailed).

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access cyclofenil, but obtained significance only for doses of >0-5 mg cyclofenil/kg. Oestradiol (20 µg/kg) did not produce any significant changes after 1 h, but after 24 h progesterone receptor concentrations were increased, the levels being similar to those achieved by 10 mg cyclofenil/kg and less than those produced by 50 mg cyclofenil/kg. Oestradiol benzoate did not significantly change progesterone receptor levels when examined 24 h after administration. The dissociation constants were not significantly changed by any of the treatments.

Biological activity By 24 h after cyclofenil treatment of the 3-week-ovariectomized rats, there was an apparent dose-related increase in uterine weight. This was accompanied by increases in the amount of uterine luminal fluid at doses of 5 mg/kg and above (Table 3). Serum LH values were unchanged but there was a dose-related increase in serum FSH concentrations. Prolactin measured in the serum of rats receiving 0-5 and 50 mg cyclofenil/kg was raised to concentrations between those achieved by oestradiol (72 ug/kg; 7-96 ± 2-88 ng/ml, = 6; < 0-05) and oestradiol benzoate (100 µg/kg; 20-28 ± 9-2 ng/ml, = 4, < 0-05). The intermediate dose of cyclofenil (10 mg/kg) failed to change serum prolactin concentrations.

Table 3. The effects of cyclofenil treatment of adult rats ovariectomized 3 weeks earlier Cyclofenil Uterine wt Uterine luminal Serum LH Serum FSH Serum prolactin (mg/kg) (mg) fluid (mg) (ng/ml) (ng/ml) (ng/ml) 0 (control) 119 ±6 (19) 3±0 6(19) 400 + 21 (20) 595 ± 39 (20) 2-6 ±0-72 (12) 0-5 137 ±5* (18) 11 2±0 9(18) 483 ±36 (14) 703 ± 33* (12) 14-96 ±4-28** (8) 1-0 130 + 4(15) 11 8±0 7(15) 456 ±33 (16) 753 ± 38* (6) 5-0 163 ±8" (12) 31 8 + 3 7** (12) 454 ± 43 (9) 826 ± 88* (6) 10 169 ± 13** (11) 26 6±4 6** (11) 434 ±25 (15) 716 ±56* (12) 3-28 + 0-36(8) 50 185 ± 12* (8) 65 5±5 7** (5) 359 ± 52 (6) 826+ 51* (6) 15-2 ±7-48* (6)

Rats were killed 24 h after treatment. Values are mean ± s.e.m. for the no. of observations indicated in parentheses. Values significantly different from the control value, *P < 0-05, **P < 0-01 (Mann-Whitney U test, two-tailed).

To ascertain the degree of oestrogenicity, the biological activity of cyclofenil was compared with that of a high dose of oestrogen, administered as oestradiol-17ß or the longer acting oestradiol-17ß benzoate. Oestradiol-17ß had no significant effects upon uterine wet weight 24 or 48 h after treatment (Table 4): the weight of the uterine luminal fluid was increased after 24 h but this was lost by 48 h. The weight of the uterus had not changed 24 h after oestradiol benzoate. However, by 48 h uterine weight had nearly doubled and the fluid content of the uterine lumen was increased 42-fold. Cyclofenil was more uterotrophic than oestradiol-17ß or oestradiol benzoate when the uterine wet weights were compared after 24 h but the changes in luminal fluid were similar. Uterotrophic activity was maintained at 48 h by cyclofenil and oestradiol benzoate treatment, but not by oestradiol, although the increase in luminal fluid at this time was not as great as that produced by oestradiol benzoate. Oestradiol decreased serum LH concentrations at 24 and 48 h in contrast to the lack of activity upon uterine weight, but oestradiol benzoate had an even greater effect. Cyclofenil did not produce any changes in serum LH concentration after 24 h but by 48 h LH concentrations were similar to those found in oestradiol benzoate-treated rats. Serum FSH values were increased by the treatments, with cyclofenil and oestradiol being more potent than oestradiol benzoate. A behavioural pattern which is activated by oestrogen is the induction of sexual receptivity (lordosis) in the ovariectomized rat. Sexual receptivity was absent in control ovariectomized rats and in rats treated with cyclofenil. After progesterone the cyclofenil treated rats displayed a high

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access Table 4. A comparison of the effects of cyclofenil, oestradiol-17ß and oestradiol-17ß benzoate treatment of adult ovariectomized rats Uterus Gonadotrophins Time killed Wet weight Luminal fluid LH FSH Treatment (h) (mg) (mg) (ng/ml) (ng/ml) Control 119±6(19) 8-3 + 0-6(19) 416 ±34 (9) 485 + 37(9) Cyclofenil (50 mg/kg) 24 185 ± 12**(8) 65-5 ±5-7** (5) 395 + 52 (6) 826+ 51* (6) 48 193 ± 10** (4) 117+ 28** (4) 146 ± 8** (4) 872 + 45** (4) Oestradiol (72 Mg/kg) 24 148 ±7 (5) 64-8+ 15** (5) 290 ± 36* (5) 1066 ±97** (5) 48 139 ± 10(5) 29-2 + 5-3(5) 258 ± 11* (5) 899+ 101* (4) Oestradiol benzoate 24 125 ± 3 (4) 69-5+ 22** (4) 185 ±39** (4) 522 + 68 (4) (100 Mg/kg) 48 224 + 7** (6) 332 ± 18** (6) 166+ 24** (4) 652 + 52** (4)

Values are mean ± s.e.m. for the no. of determinations in parentheses. Significantly different from the control value, *P < 0-05, **P < 0-01 (Mann-Whitney U test, two-tailed).

level of sexual receptivity (LQ = 89%) which was not significantly different from that produced by oestradiol-17 ß benzoate (LQ = 100%). The number of animals per treatment group was 5-9. The ability of cyclofenil to antagonize the effects of oestrogen was investigated in adult rats, ovariectomized 1 week earlier or in immature 25-day-old female rats. The 50 mg/kg dose of cyclofenil was chosen because it is known to occupy the oestrogen receptors of the uterus, brain and pituitary gland. The results are presented in Table 5. In ovariectomized adult rats, cyclofenil was as uterotrophic as oestradiol-17ß benzoate and the combinations of the two drugs did not lead to any significant changes when compared to those with either drug alone. In the immature female rat cyclofenil was less uterotrophic than oestradiol-17ß benzoate and the combination of the two drugs gave uterine weights similar to those after cyclofenil alone. Cyclofenil clearly antagonized the effects of oestradiol- 17ß benzoate upon the accumulation of luminal fluid of the immature and mature rat uterus, the weights of the luminal fluid after combination of the two drugs being similar to the effects of cyclofenil alone. In mature ovariectomized rats cyclofenil acted as an oestrogen to decrease the serum concentration of LH: no antagonism was seen when the two drugs were administered together. In immature rats only cyclofenil produced a significant decrease in LH concentration. Multiple doses of cyclofenil did not change serum FSH concentration. Oestradiol benzoate decreased serum FSH concentrations, the decrease being partly antagonized by cyclofenil. Serum concentrations of FSH in the immature rat were unchanged by all of the treatments.

Table 5. The separate and combined effects of cyclofenil and oestradiol benzoate on immature and ovariectomized adult rats

Ovariectomized adults Immature

Uterine Uterine Uterine wt luminal LH FSH Uterine wt luminal LH FSH Treatment^ (mg) fluid (mg) (ng/ml) (ng/ml) (mg) fluid (mg) (ng/ml) (ng/ml)

Control, arachis oil 133+ 14 9±0-6 171 ± 16 657± 115 41 ± 12 5-l± 1-9 48±6 196+115 Oestradiol benozate 341 +20+t 816 ± 97tt 62±9tt 312±9t 132+ 10++ 98±36t 37 ±4 99 + 35 (28 Mg/kg) Cyclofenil (50 mg/kg) 342±litt 401 ±72tt 45±13tt 619+75 99±9t 10-3+1-2+ 27±6t 83 + 24 Oestradiol benzoate + 303 ±16* 414 + 50" 50±3 480 ±32* 82 ±4*· 9-7 ±1-6* 38 + 4 107 ±5 cyclofenil

i; Given daily for 3 days: the rats were killed 24 h after the last dose. Values are mean + s.e.m. for 5 rats/group. Values significantly different from the control value. *P < 0-05. **P < 0-01. Values significantly different from the control value for the oestradiol benzoate-treated rats. fP <0-05. t+ < 001 '(Mann-Whitney U test).

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access Discussion

The results demonstrate that cyclofenil will react directly with the oestrogen receptor system of the brain, pituitary gland and uterus. In vitro, cyclofenil produced a concentration-related decrease of PHloestradiol binding to the uterine cytoplasmic oestrogen receptor. The inhibition of the oestradiol binding was competitive and cyclofenil bound to the oestrogen receptor with high affinity, Kd] = 0-94 IO-8 mol/1. The affinity of cyclofenil for the oestrogen receptor is of the same order as that reported by Geynet, Millet, Truong & Baulieu (1972) and is similar to the affinities for the oestrogen receptor determined for the oestrogen antagonists clomiphene and tamoxifen, but is lower than that determined for oestradiol and diethylstilboestrol (Geynet et al, 1972; Ginsburg et al, 1977; Wakeling & Slater, 1980). The in-vivo effects of cyclofenil upon the cytoplasmic oestrogen receptor concentrations were complicated and did not follow a simple dose-related depletion as has been observed for the non-steroidal oestrogen antagonists (Jordan, Dix, Naylor, Prestwich & Rowsby, 1978; Kurl & Morris, 1978; Katzenellenbogen et al, 1980). In addition, the receptor changes induced by cyclofenil were similar in brain and peripheral target tissue. Low doses of clomiphene and tamoxifen only change the receptor concentrations in peripheral tissues, much higher doses being required to affect the brain oestrogen receptors (Kurl & Morris, 1978). Therefore, if the biological activity of these drugs is similar the exclusion of a drug from the brain is probably not an important feature with respect to the induction of ovulation by these drugs. Both the high and low dose of cyclofenil depleted the cytoplasmic oestrogen receptor concentration, although depletion was not observed following the intermediate doses. These predominantly unchanged receptor concentrations considered in isolation appear to indicate the absence of effects upon the oestrogen receptors, even though the biological data indicate otherwise. It is now well established that the cytoplasmic oestrogen receptor status of a tissue is the net result of oestrogen acting upon the mechanisms responsible for depletion, recycling and resynthesis of receptor (Clark & Peck, 1979; Katzenellenbogen et al, 1980). Using these concepts, it is possible to explain the present results. After the initial combination of cyclofenil with the receptor, the receptor complex is translocated to the nucleus. By 24 h after administration of the smaller doses, cyclofenil has been cleared from the body as indicated by the absence of changes in nuclear receptor concentrations. Therefore it would appear that cytoplasmic receptor concentrations remain low, not because of occupation and/or depletion, but because of the lack of receptor resynthesis. The absence of protein synthesis is supported by the observation that synthesis of progestagen receptor, which is an oestrogen-dependent phenomenon, does not occur after the low dose of cyclofenil. Increased protein synthesis is indicated after the higher doses of cyclofenil (10 and 50 mg/kg) by the increases in progestagen receptor concentrations. It is probable that the oestrogen receptor proteins are also synthesized, as indicated by the high concentrations of oestrogen receptor after 10 mg cyclofenil/kg. Nuclear receptors after this dose were not increased. The increase of receptors after 50 mg cyclofenil/kg corresponds with a fall in cytoplasmic receptor concentration, which suggests that cyclofenil is still circulating in the blood and is available to interact with free oestrogen receptors in the target cells. Cyclofenil increased the uterine progesterone receptor concentration and in this respect is similar to the non-steroidal oestrogen antagonists such as tamoxifen and nafoxidine-like compounds (Jordan & Dix, 1979; Allen & Jordan, 1980). However, unlike the oestrogen antagonists, the concentrations of progesterone receptor achieved by cyclofenil were greater than with oestradiol or oestradiol benzoate. This result reflects the marked oestrogenicity of cyclofenil which was also demonstrated by the increases in uterine weight, uterine luminal fluid volume, sexual receptivity, serum FSH and prolactin concentrations, and decreases in serum LH concentration. The experiments confirm and extend the work of previous publications which suggested that cyclofenil was a partial oestrogen agonist (Einer-Jensen, 1968; Carlborg, 1970).

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access However, the pharmacology of cyclofenil is complicated; in the ovariectomized adult rat a single dose of cyclofenil will produce changes in uterine weight and uterine cytoplasmic progesterone receptors which are greater than, or equivalent to, the changes which can be produced by a large dose of oestrogen. The uterotrophic changes after multiple doses of cyclofenil were also similar to the changes produced by oestrogen. The data would suggest that cyclofenil is a full agonist in relation to uterine weight. This conclusion differs from that of Einer-Jensen (1968) who studied mice. The activity of the non-steroidal oestrogens differs in the mouse and rat; tamoxifen, for example, is a partial agonist in the rat and a full agonist in the mouse (Furr et al, 1979). Cyclofenil acted more quickly on the uterus than did the oestrogens, probably because of the different pharmacokinetics of the compounds. Oestradiol-17ß is. active for only a short period as it is rapidly cleared from the body, oestradiol-17ß benzoate has to be hydrolysed to oestradiol-17ß before it is active. The results indicate that cyclofenil is active immediately and the slow clearance rate would account for the prolonged biological activity (Larsson, 1965). LH secretion in adult ovariectomized rats was inhibited by both cyclofenil and oestrogen. Cyclofenil also inhibited LH secretion in the immature female rat but oestrogen was without effect. In the ovariectomized female rat serum FSH concentrations were increased by a single dose of either cyclofenil or oestrogen. Multiple doses of oestradiol-17ß benzoate suppressed serum FSH while cyclofenil did not produce any significant change. The latter observation may reflect a transition from a stimulatory to an inhibitory action upon FSH secretion. Taubert & Baier (1969) reported similar changes in gonadotrophins, measured by bioassay, when cyclofenil was administered to oestrogen-progestagen-treated rats, whereas in intact rats cyclofenil increased both LH and FSH (Seki, Seki, Takayanagi, Yoshihara & Maeda, 1972). The ability of non-steroidal oestrogens to alter gonadotrophin secretion is well documented. We have previously reported that en-clomiphene will also inhibit serum FSH and LH secretion (Bowman, Leake, Miller & Morris, 1981). However, increases in serum FSH were not seen after clomiphene, and the LH changes were not so marked. Clomiphene and tamoxifen will also increase plasma prolactin concentrations in the ovariectomized rat (Bowman et al, 1981, unpublished); 24 h after cyclofenil, increased serum prolactin concentrations were detected for all except the intermediate dose. The reasons for this biphasic effect are not clear. High doses of clomiphene do not increase serum prolactin concentrations until 16 days after administration, whereas a low dose is active after 2 days; the effects of cyclofenil may also be time- as well as dose-related (Bowman et al, 1981). Cyclofenil has been reported to induce sexual receptivity in the bitch and the mare (Einer-Jensen, 1968). In ovariectomized rats, however, unlike the effect of oestradiol-17ß benzoate, sexual receptivity was not detected unless the rats were treated with progesterone. This would indicate that cyclofenil had induced the synthesis of hypothalamic as well as uterine progesterone receptors, as the ability of progesterone to affect sexual behaviour is related to the induction of hypothalamic progesterone receptors by oestrogen (Moguilewsky & Raynaud, 1979). Other non-steroidal oestrogens which are partial agonists upon the uterus, e.g. en-clomiphene, tamoxifen and CI 628, are not able to induce sexual receptivity when administered under similar conditions (Feder, Landau & Walker, 1979; Roy, Schmit, McEwen & Wade, 1979; Bowman et al, 1981). Oestrogen antagonism by cyclofenil has been demonstrated upon the chick oviduct (Einer-Jensen, 1968) and the rat uterus (Watnik & Neri, 1968). Watnik & Neri (1968) demonstrated that 12 mg cyclofenil administered orally for 3 days would antagonize the uterotrophic effects of stilboestrol in the ovariectomized rat. The present study investigated the possible antagonism of oestradiol-17ß benzoate by cyclofenil in immature and adult ovariectomized rats; antagonism of the uterine weight increase was observed in the immature rats but not in the adult rats. A pronounced antagonism of oestrogen-induced increases in uterine fluid volume was demonstrated for rats of both ages and with a low dose of 5 mg/kg per day for 3 days (data not given). The oestrogen-induced accumulation of uterine luminal fluid is therefore

Downloaded from Bioscientifica.com at 09/27/2021 08:32:47PM via free access more sensitive to the oestrogen antagonists than is uterine weight, as shown by Bowman et al (1981). In conclusion we have demonstrated that cyclofenil. a non-steroidal compound, will interact with the oestrogen receptor system through which its appreciable biological activity is probably mediated. Most non-steroidal oestrogens can be classified as agonists or partial agonists. The oestrogenicity of the partial agonists is usually small and the antagonist activity predominates (Furr et al, 1979; Clark & Peck, 1979). Cyclofenil may be unique in that, in the rat at least, it is the agonist activity that is appreciable with antagonist activity being undetectable in some target tissues. It is therefore questionable whether the activity of cyclofenil in man can be attributed to antioestrogenicity. Indeed, there are several clinical reports which comment upon the oestrogenicity of cyclofenil upon, for example, the cervical mucus and the male breast (Terner, 1966; Sato et al, 1969; Murray & Osmond-Clarke, 1971). Thus the activity of cyclofenil in the treatment of female infertility is probably attributable to the oestrogenicity rather than the antioestrogenicity of the cyclofenil molecule. We gratefully acknowledge the financial support of Birthright, the gift of cyclofenil from Roussel Laboratories, Wembley, U.K., and Ferrosan, Malmö, Sweden, and the NIAMDD radioimmunoassay kits. A.L. was supported by the S.R.C.

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