The effects of the antiestrogen CI-628 on sexual behavior activated by androgen or estrogen in quail

This series of experiments sought to determine whether conversion of androgen to estrogen is important in the activation of male sexual behavior in quail by seeing if an antiestrogen will block androgen stimulated copulation in this species. Experiment I compared the ability of two antiestrogens, MER-25 (5 mg/day) and CI-628 (2 mg/day), to block estrogen stimulated characteristics in female quail. Both treatments greatly reduced oviduct growth in “photically castrated” females given estradiol benzoate (EB, 50 μg/day), but only CI-628 reduced receptivity in these birds. In Experiment II surgically castrated males given 50 μg/day EB together with 2 mg/day CI-628 were much less receptive than castrated males given EB alone, and in addition copulated in fewer tests. In Experiments III, IV, and V, castrated males given testosterone propionate (TP) together with CI-628 were compared with males given TP alone. The ability of CI-628 to suppress TP-stimulated copulation increased with increasing CI/TP dosage ratio, and at the highest ratio (4:1), CI-628 effectively blocked copulation in five out of seven birds. Those birds that did copulate did so in fewer tests and performed fewer cloacal contact movements. CI-628 had no antiandrogenic effects in these experiments. These results suggest that estrogens may be important active metabolites of testosterone with respect to quail copulation.     

The effect of the antiestrogen CI-628 on androgen-induced aggressive behavior in castrated male mice

This study was conducted to determine whether or not the antiestrogen CI-628 would block testosterone-maintained fighting in castrated male mice. TO strain adult male mice were castrated and injected s.c. every day for 14 days with either (1) 75 μg testosterone or (2) 75 μg testosterone and 1 mg CI-628, and in addition 1 mg of CI-628 6 hr prior to each injection of antiestrogen and androgen. Vehicle-injected, castrated, and CI-628-injected animals were employed as controls. Testosterone-maintained intermale aggressive behavior was blocked by the antiestrogen CI-628. This study provides support for the hypothesis that testosterone exerts its effects on the central nervous elements involved in the control of aggressive behavior by its aromatization to estrogenic metabolites.     

Blockade of the preovulatory release of gonadotropins and prolactin by spinal transections in female rats

The effect of spinal transections on the preovulatory release of gonadotropins and PRL was investigated in female rats. A preovulatory rise in serum LH, FSH and PRL and subsequent ovulation were prevented by complete spinal transections (CST) at high thoracic levels (T3-T7), but not at low thoracic and lumbar levels (T8-L5), performed at 1000-1230 h on proestrus. Norepinephrine (NE) concentrations in the preoptic-anterior hypothalamic area at 1700-1800 h on proestrus were also significantly reduced by CST at high thoracic levels, but not at lumbar levels. Either electrochemical stimulation of the suprachiasmatic part of the preoptic area or NE injection into the third ventricle at 1400-1500 h on proestrus restored ovulation in animals with CST at high thoracic levels. Animals with CST at lumbar levels exhibited relatively regular 4-day cycles, but those with CST at high thoracic levels showed prolonged periods of diestrous (8-20 days) before they resumed cyclicity. In the case of partial transections, bilateral transections of the lateral columns, but not transections of the dorsal or medial columns, of the spinal cord at T4-T5 significantly blocked the preovulatory gonadotropin release and the occurrence of ovulation. Unilateral transections of the lateral columns of the spinal cord or unilateral electrolytic lesions of the ventrolateral part of the medulla oblongata (VLMO) failed to block ovulation. When combinations of them were performed ipsilaterally, ovulation occurred, but when they were performed contralaterally, the incidence of ovulation was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of sexual receptivity in the female lizard, Anolis carolinensis: Effects of estrogen and the antiestrogen CI-628

Daily behavioral testing revealed that there is a latency period of at least 48 hr from the administration of a single injection of estradiol benzoate (EB) to the first significant increase in female sexual receptivity in the ovariectomized female lizard, Anolis carolinensis. This latency period did not vary with dosage of EB used in these experiments (i.e., 0.8, 1.4, and 4.0 μg) nor with method of injection (subcutaneous vs intraperitoneal for dose of 1.4 μg EB). Following a single EB injection, female sexual receptivity increased after the 48-hr latency period, reached an observed peak from Day 3 to Day 6, and thereafter declined to pretreatment levels by Day 19. Although both 1.4 and 4.0 μg of EB produced higher levels of female sexual receptivity than did treatment with 0.8 μg of EB, results obtained with 4.0 μg EB did not differ from those obtained with 1.4 μg EB. Administration of the nonsteroidal antiestrogen CI-628 (80 μg) at either 4 or 24 hr following a single subcutaneous injection of 1.4 μg EB significantly reduced subsequent female sexual receptivity. These results suggest that there is a critical length of time during which estrogen must act on the brain and support the concept of an estrogen “maintenance” effect during this priming period.     

Effects of estradiol on the prolactin surges and the feedback mechanisms of gonadotropins in pseudopregnant rats

The influences of estradiol on the prolactin (PRL) surges and on the secretion of gonadotropins (LH and FSH) were investigated in the pseudopregnancy (PSP) of acutely ovariectomized rats. The four following experimental groups were prepared: 1) intact PSP as a control, 2) ovariectomy was performed on day 0 of PSP (OVX), 3) a Silastic tube containing estradiol was implanted for day 1-4 into the OVX rats (OVX-E 1-4), and 4) the Silastic tube was implanted for day 5-8 by the same manner into the OVX rats (OVX-E 5-8). In the OVX group nocturnal (N) PRL surges were observed at 0500 h on days 4, 8 and 12 examined, and increased secretions of LH and FSH were noted. In the OVX-E 1-4 group, the N PRL surge was suppressed on day 4, and the suppressed N PRL surge did not occur on day 8, after the removal of the implanted tubes. Diurnal (D) PRL surges with LH surges were observed at 1700 h on day 4 in these rats. Similarly, more remarkable results were obtained on days 8 and 12 in the OVX-E 5-8 group than in the OVX-E 1-4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of MCF 7 breast cancer cell growth inhibition by the antiestrogen nitromifene (CI 628) and selected metabolites

Besides undergoing O-demethylation in vivo, the triarylethylene antiestrogen nitromifene [1-(4-(2-pyrrolidinylethoxy)phenyl)-1-(4-methoxy)-phenyl-2-phenyl- 2- nitroethene, 1] undergoes biotransformation via nitroreduction, ethene bond cleavage, and pyrrolidine ring oxidation affording ketone metabolites 2 and 3 and a lactam metabolite 4. Estrogen receptor (ER) affinities of 1, 2, and 4 were, in turn, 1.7, 0.1, and 3.8% that of estradiol in MCF 7 human breast cancer cells, and these compounds inhibited by 50% the proliferation of MCF 7 cells at respective concentrations of 1.1, 5.6, and 2.0 microM. The inhibitory effect of 4 was fully reversible by estradiol, but that of 2 was only partially reversible. Also 3, which did not interact with ER, inhibited proliferation by 44% at a concentration of 10 microM. These results suggested that in contrast to 4, the effects of 2 and 3 were due in part to interaction with sites distinct from ER. Antiestrogen binding sites and calmodulin have been suggested to mediate antiproliferative effects of drugs. Interaction of ligands with the former sites has been proposed to antagonize the growth promoting effect of histamine. Although 2 and 3 had high affinities for these sites, their inhibitory effects on MCF 7 cell growth were largely unaffected by the presence of histidine, the source of intracellular histamine. Thus, the relationship between antiestrogen binding site affinity and antiproliferative effects of 2 and 3 was not clarified. In contrast, MCF 7 cell growth suppression potencies paralleled calmodulin antagonist potencies of 1 and 2 suggesting that interaction of 1 and 2 with calmodulin may contribute to their anticancer effects.     

Site of action for endotoxin-induced cortisol release in the suppression of preovulatory luteinizing hormone surges

The study was conducted to identify the mechanisms of endotoxin/cortisol action in the suppression of preovulatory LH surges in heifers infused with Escherichia coli (E. coli ) endotoxin. The hypotheses tested were that 1) endotoxin stimulates the release of progesterone, possibly from the adrenal leading to the LH blockade; 2) cortisol released in response to endotoxin infusion blocks the synthesis of estradiol at the ovarian level, culminating in a failure of the LH surge. Eight Holstein heifers were given two injections of prostaglandin F(2alpha) (PG), 11 d apart, to synchronize estrus. Starting from 25 h after the second injection of PG (PG-2), the uterus of each heifer was infused either with 5 ml of pyrogen-free water (control, n = 3) or with E. coli endotoxin (5 mug/kg of body weight) in 5 ml of pyrogen-free water (treated, n = 5), once every 6 h for 10 treatments. Blood samples were obtained every 15 min for 1 h before infusion and again 2 h after each infusion, then hourly until 1 h before the next infusion. After the tenth infusion, blood was collected daily until estrus. Serum progesterone concentrations remained at baseline values (< 1 ng/ml) in control and treated heifers. The total amount of progesterone measured starting 24 to 84 h after PG-2 injection was not different between control and treated heifers (P 0.05). In the control heifers, serum estradiol concentrations remained basal (< 10 pg/ml) until 4 h before the LH surge. Serum estradiol concentrations increased to 20 +/- 5.6 pg/ml, 4 h before the LH surge in control heifers (LH surge occurred 60 to 66 h after the PG-2 injection). There were no changes in serum estradiol concentrations in treated heifers during the sampling period, and the concentrations remained < 10 pg/ml. The total amount of estradiol measured in control heifers was higher (P < 0.05) than in treated heifers. The results if this study suggest that increases in cortisol concentrations after the infusion of endotoxin might block the synthesis of estradiol at the ovarian level, resulting in the failure of a preovulatory LH surge to occur.     

Direct inhibition of testosterone synthesis in rat testis interstitial cells by ethanol: possible sites of action

The present in vitro studies using interstitial cells of adult rat testes demonstrated that ethanol inhibits LH- and 8-bromo-cyclic AMP-stimulated testosterone synthesis, pregnenolone- and progesterone-stimulated testosterone synthesis, and basal testosterone synthesis. However, the patterns of inhibition following exposure to 0.22 to 880 or 1100 mM ethanol were different. In general, the inhibition curves for LH-, 8-bromo-cyclic AMP-, pregnenolone- and progesterone-stimulated testosterone synthesis were biphasic, with a gradual slope from 0.22 to 220 mM ethanol, and a sharper slope with concentrations of ethanol greater than 220 mM. Basal testosterone synthesis was reduced only to 74% of control with ethanol concentrations up to 44 mM, and higher concentrations of ethanol reduced testosterone synthesis no further. The effect of ethanol on Lh-stimulated cyclic AMP accumulation showed an even different pattern: some of the lower concentrations of ethanol inhibited cyclic AMP accumulation, while higher levels of ethanol progressively increased cyclic AMP accumulation. These studies demonstrate that isolated interstitial cells are highly sensitive to the direct effects of ethanol; they also suggest that the principle site of ethanol inhibition may be at the level of the smooth endoplasmic reticulum where progesterone is converted to testosterone.     

Corticotropin-releasing factor inhibition of substance P-induced vascular leakage in rats: possible sites of action.

Substance P (SP), 40 micrograms/kg SC, induced protein leakage in the skin, muscle, trachea and esophagus of the anesthetized rat as measured by Monastral blue B labeling of small blood vessels. CRF, 30 micrograms/kg SC, injected 30 min before SP, decreased the SP-induced dye leakage. To locate where CRF might act, autoradiographic studies of [125I]-CRF binding to esophageal segments were conducted and displaceable binding of [125I]-CRF to submucosal elements in the esophageal epithelium were revealed, suggesting that CRF acts on selective sites to reduce vascular leakage.     

The possible role of calmodulin in the inhibition of prolactin secretion by dopaminergic antagonists

Several previous reports have indicated that a number of dopaminergic antagonists paradoxically inhibit prolactin secretion at micromolar concentrations. It is well known that some of these drugs, including pimozide and the phenothiazines, are inhibitors of calmodulin activity. Here we report that micromolar concentrations of several dopaminergic antagonists inhibit prolactin secretion from isolated rat anterior pituitary cells and calmodulin activity (calmodulin-activated cyclic GMP phosphodiesterase). Inhibition of calmodulin activity may thus, at least partially, explain the inhibitory effect of these drugs on prolactin secretion.     

Suppression of the prolactin surges in the pseudopregnant rat by urethane anesthesia

In pseudopregnancy of the rat prolactin (PRL) is released in the form of twice daily surges (nocturnal and diurnal surges). An attempt was made to examine the effects of urethane anesthesia on PRL surges during pseudopregnancy of the rat. In a preliminary study, using the continuous blood sampling method, the nocturnal PRL surge was completely blocked when urethane (1.0g/kg BW) was administered at 0:00 hr. Urethane (1.0g/kg BW) was injected at 0:00 or 12:00 hr, and serum and pituitary PRL concentrations were measured at 6:00 or 18:00 hr, respectively, to study the effects of urethane on nocturnal or diurnal PRL surges. There were no serum PRL surges during either the nocturnal or diurnal periods following urethane injection. The experiment examining pituitary PRL concentration at 6:00 or 18:00 hr confirmed that urethane (1.0g/kg) anesthesia suppressed the release of PRL surge from the pituitary.     

Inhibition by prolactin of gonadotrophin-induced cyclic AMP accumulation in the preovulatory ovary

We investigated whether prolactin acts at the ovarian level by interfering with the accumulation of gonadotrophin-induced ovarian cyclic AMP. Mouse ovaries were incubated with hCG and varying doses of prolactin. At the end of the incubation, the cyclic AMP which accumulated in the tissue + medium was measured. In ovaries devoid of corpora lutea, a significant inverse correlation (r = -0.93, P less than 0.05) was obtained between the doses of prolactin (0.1-25.6 micrograms ovine prolactin) and hCG-induced accumulation of ovarian cyclic AMP. In the presence of the phosphodiesterase inhibitor IBMX, however, the same doses of prolactin failed to exhibit any restricting influence on the accumulation of cyclic AMP. In luteinized ovaries, the same doses of prolactin in the absence of IBMX did not inhibit the hCG-induced cyclic AMP accumulation.     

Absorption of zinc and retention of calcium: Dose-dependent inhibition by phytate

The dose-dependent inhibitory effect of sodium phytate (myo-inositol-hexaphosphate) on absorption of zinc and retention of calcium was studied in man. No systematic study of this dose-response effect has been reported to this time. Forty subjects were served meals containing white wheat rolls without/with additions of phytate. Ten subjects were given test meals containing one or two of the studied levels of phytate and in addition all subjects were served meals to which no phytate was added. The zinc content was 3.1 mg (47 micromol) and the calcium content 266 mg (6.6 mmol). The rolls were labelled extrinsically with radioisotopes, 65Zn and 47Ca, and whole-body retention of both minerals was measured. Totally 105 meals were served, 36 meals in which no phytate was added and 9-10 meals on each level of phytate. The zinc absorption in meals to which either 0, 25, 50, 75, 100, 140, 175 or 250 mg of phytate-P (0, 134, 269, 403, 538, 753, 941 or 1344 micromol phytate) had been added was 22%, 16%, 14%, 11%, 7%, 7%, 7% and 6%, respectively (mean values). The addition of 50 mg phytate-P or more significantly decreased zinc absorption (p=0.01) as compared to absorption from the test meals with no added phytate. The calcium retention at day 7 in the same meals was 31%, 28%, 27%, 26%, 22%, 19%, 14% and 11% (mean values). The addition of 100 mg phytate-P or more significantly decreased calcium retention (p=0.03) compared to the test meals with no added phytate. It was concluded that the inhibitory effect of phytate on the absorption of zinc and the retention of calcium was dose dependent.     

DNA repair inhibition: a possible mechanism of action of co-carcinogens

Co-carcinogens with a wide diversity of chemical structure have the common property of inhibiting DNA repair replication in normal human lymphocytes. This evidence suggests that inhibition of DNA repair may be an important factor in the mechanism of action of co-carcinogens.     

Down regulation of prolactin receptors in the hamster ovary by the preovulatory gonadotropin surge

Binding of 125I-prolactin (Prl) to hamster ovarian homogenates was found to decrease markedly at the time of the preovulatory gonadotropin surge (PGS). Saturation analysis revealed that the decrease was due to a reduction in the number of available Prl receptors and not due to a change in binding affinity. Loss of Prl receptors following the PGS was not affected by treatment with ergocryptine to block the release of pituitary Prl, indicating that the reduction in the number of available Prl receptors was not due to increased occupancy by endogenous Prl. Loss of Prl receptors was prevented by treatment with phenobarbital (Phen) to block the normal luteinizing hormone (LH)/follicle-stimulating hormone (FSH) surge; whereas, an injection of 50 micrograms of LH or 50 micrograms FSH (but not 100 micrograms Prl) induced a marked decrease in Prl receptors in Phen-treated hamsters. To determine whether Prl receptor loss induced by 50 micrograms FSH might be due to LH contamination, Phen-treated hamsters were injected with minimal ovulatory doses of LH and FSH. Injection of 5 micrograms or 2.5 micrograms LH induced a loss of Prl receptors in 90% and 70% of Phen-treated hamsters, respectively. In contrast, injection of 5 micrograms or 2.5 micrograms FSH induced a loss of Prl receptors in 0% and 20% of Phen-treated hamsters, respectively. These results indicate that the PGS causes an acute heterologous down regulation of ovarian Prl receptors and suggest that this down regulation may be due principally to the action of LH.     

Changes in follicular steroidogenic enzymes following the preovulatory surge of gonadotropins and experimentally-induced atresia

Atresia that is induced experimentally and the preovulatory surge of gonadotropins stimulate similar changes in follicular steroidogenesis in the rat, i.e., both enhance production of progesterone and reduce production of androgen and 17 beta-estradiol. In this study, mature cycling rats were either stimulated with human chorionic gonadotropin (hCG) or atresia was induced by blocking the proestrous surge of gonadotropins through the use of pentobarbitone or hypophysectomy. Changes in activity of C17,20-lyase (lyase) and 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) were estimated from homogenates of 10-15 Graafian follicles by evaluating conversion of precursors to products that were separated and quantified by high performance liquid chromatography (HPLC). Within 3 h of administration to proestrous rats, hCG reduced follicular lyase activity (pmole androstenedione produced per mg protein during 30 min incubation) from (mean +/- SEM) 221.3 +/- 24.2 to 120.2 +/- 30.4, and to 8.5 +/- 0.1 after 9 h. By contrast, 20 alpha SDH activity increased somewhat after hCG stimulation. Similar changes were observed after follicular atresia was induced, with hypophysectomy causing the most striking changes. Lyase was reduced to 60% within 6 h after the operation, and to 2% within 24 h. Activity of 20 alpha SDH was doubled within 6 h of hypophysectomy and remained high even 24 h later. Thus, in preovulatory rat follicles, luteinizing hormone (LH)/hCG reduces lyase activity and similar changes occur in such follicles undergoing atresia. There was no clear correlation between 20 alpha SDH and lyase activities; our results did not support the argument that 20 alpha SDH products regulate lyase following the ovulatory stimulus and atresia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estradiol induces and progesterone inhibits the preovulatory surges of luteinizing hormone and follicle-stimulating hormone in heifers

Objectives were to determine: 1) whether estradiol, given via implants in amounts to stimulate a proestrus increase, induces preovulatory-like luteinizing hormone (LH) and follicle-stimulating hormone (FSH) surges; and 2) whether progesterone, given via infusion in amounts to simulate concentrations found in blood during the luteal phase of the estrous cycle, inhibits gonadotropin surges. All heifers were in the luteal phase of an estrous cycle when ovariectomized. Replacement therapy with estradiol and progesterone was started immediately after ovariectomy to mimic luteal phase concentrations of these steroids. Average estradiol (pg/ml) and progesterone (ng/ml) resulting from this replacement were 2.5 and 6.2 respectively; these values were similar (P greater than 0.05) to those on the day before ovariectomy (2.3 and 7.2, respectively). Nevertheless, basal concentrations of LH and FSH increased from 0.7 and 43 ng/ml before ovariectomy to 2.6 and 96 ng/ml, respectively, 24 h after ovariectomy. This may indicate that other ovarian factors are required to maintain low baselines of LH and FSH. Beginning 24 h after ovariectomy, replacement of steroids were adjusted as follows: 1) progesterone infusion was terminated and 2 additional estradiol implants were given every 12 h for 36 h (n = 5); 2) progesterone infusion was maintained and 2 additional estradiol implants were given every 12 h for 36 h (n = 3); or 3) progesterone infusion was terminated and 2 additional empty implants were given every 12 h for 36 h (n = 6). When estradiol implants were given every 12 h for 36 h, estradiol levels increased in plasma to 5 to 7 pg/ml, which resembles the increase in estradiol that occurs at proestrus. After ending progesterone infusion, levels of progesterone in plasma decreased to less than 1 ng/ml by 8 h. Preovulatory-like LH and FSH surges were induced only when progesterone infusion was stopped and additional estradiol implants were given. These surges were synchronous, occurring 61.8 +/- 0.4 h (mean +/- SE) after ending infusion of progesterone. We conclude that estradiol, at concentrations which simulate those found during proestrus, induces preovulatory-like LH and FSH surges in heifers and that progesterone, at concentrations found during the luteal phase of the estrous cycle, inhibits estradiol-induced gonadotropin surges. Furthermore, ovarian factors other than estradiol and progesterone may be required to maintain basal concentrations of LH and FSH in heifers.     

Pituitary site of action of endothelin: selective inhibition of prolactin release in vitro

Endothelin-3 (ET-3) inhibited in a dose-dependent, significant fashion prolactin release from cultured anterior pituitary cells (ovariectomized female and intact male rat donors, ED50 = 5 X 10(-9) M). ET-3 in log doses ranging from 10(-11) to 10(-6) M did not alter significantly the release of luteinizing hormone, growth hormone or thyroid stimulating hormone. The inhibitory effect of ET-3 (rat, human) was specific for that molecule since ET-1 (porcine, human) was ineffective and was not due to an action on the dopamine receptor since the inhibitory action was still expressed in the presence of 100 nM domperidone. These data further suggest a role for neuropeptides of the posterior lobe in the control of lactotroph function.     

Role of estrogen receptors and antiestrogen binding sites in an early effect of antiestrogens, the inhibition of cholesterol biosynthesis

The effects of estradiol (E2), 4-hydroxy-tamoxifen (OH-Tam), and LY117018 on cholesterogenesis were investigated in two human breast cancer cell lines (MCF-7 and BT20), and in rat hepatoma (HTC) and fibroblastic (NRK-49F) cell lines. It was found that 10(-10) M E2 stimulated and 10(-8) M OH-Tam inhibited cholesterol synthesis in the estrogen-sensitive MCF-7 cell line. The OH-Tam effect occurred in less than 15 min whereas E2 only stimulated after 8 h. The inhibition of cholesterol synthesis was not reversed by E2. E2 was without effect in the HTC and estrogen-resistant BT20 cell lines whereas OH-Tam was as effective as in the MCF-7 cells. LY117018 had nearly as much effect on cholesterol synthesis as OH-Tam, in both MCF-7 and BT20 cells. Neither E2 nor OH-Tam had any effect on the NRK-49F cell line, even at micromolar concentrations. The three lines (MCF-7, BT20, HTC), whose cholesterol synthesis has been shown to be OH-Tam sensitive, appeared to contain high-affinity antiestrogen binding sites (AEBS); since the OH-Tam-resistant line (NRK) only contained low-affinity AEBS, there appears to be some relationship between OH-Tam sensitivity and high-affinity AEBS content. This suggests that the cholesterogenesis inhibition induced by antiestrogens is ER-independent and may involve AEBS. The cholesterogenesis stimulation induced by E2 occurred via a different pathway that appears to be related to the presence of ER in the cells.