Effects of Opiate Antagonists on Early Pregnancy and Pseudopregnancy in Mice
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Effects of opiate antagonists on early pregnancy and pseudopregnancy in mice G. L. Nieder and C. N. Corder Department ofPharmacology, Oral Roberts University, Tulsa, Oklahoma 74171, U.SA. Summary. Administration of naltrexone or the long-acting morphine antagonist chlornaltrexamine before infertile mating had no effect on the length of the resulting pseudopregnancy in mice. Naltrexone in doses of 10 to 200 mg/kg s.c. given on Days 2 or 3 of pregnancy showed no consistent effects on the maintenance of pregnancy. Multiple doses or intracerebroventricular administration of naltrexone also had no effect. Chronic infusion of naltrexone, provided by mini-osmotic pumps, from Day 1 of pregnancy had no effect on the incidence of pregnancy or the number of embryos implanted. These results suggest that endogenous opioids do not play a critical role in this prolactin-dependent physiological process. Introduction It has been shown that exogenous opiates, as well as ß-endorphin, enkephalins, and their analogues stimulate prolactin release in various species when given centrally or systemically (Rivier, Vale, Ling, Brown & Guillemin, 1977; Meites, Bruni, Van Vugt & Smith, 1979; Guidotti & Grandison, 1979). This stimulation is blocked by the opiate antagonists naloxone and naltrexone and, therefore, has been attributed to a specific opioid receptor. Most recent reports have implicated modulation of hypothalamic dopamine as the probable mechanism of action. Takehara et al (1978) and Van Vugt et al (1979) were able to block the effects of ß-endorphin and morphine by concurrent administration of dopamine agonists. Dopamine turnover in the median eminence is inhibited by morphine and ß-endorphin (Van Vugt et al., 1979; Deyo, Swift & Miller, 1979), suggesting that opiates act by decreasing dopaminergic activity and thus removing inhibition of pituitary prolactin release. Endogenous opioids have been implicated in the physiological prolactin response to various stimuli. Stresses due to ether (Ferland, Kledzik, Cusan & Labrie, 1978), footshock (Grandison & Guidotti, 1977), heat and immobilization (Van Vugt, Bruni & Meites, 1978) result in increased serum prolactin concentrations, an effect which is blocked partly or completely by opiate antagonists. The effect of naloxone on the prolactin surge at pro-oestrus has been studied by Muraki, Nakadate, Tokunaga, Kato & Makino (1979) who found that naloxone blocked the inhibition of the prolactin surge by morphine, but had no effect by itself. Conversely, Ieiri, Chen, Campbell & Meites (1980) totally blocked the prolactin surge with naltrexone, an effect which was reversible with morphine. A decrease in basal serum prolactin has been seen in male rats treated with naloxone (Bruni, Van Vugt, Marshall & Meites, 1977) and naltrexone (Guidotti & Grandison, 1979). Other reports have failed to demonstrate any effect of antagonists in rats or * Reprint requests to Dr C. N. Corder. t Present address: Department of Pharmacology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. 0022-4251 /82/040341 -06S02.00/0 1982 Journals of Reproduction & Fertility Ltd Downloaded from Bioscientifica.com at 10/03/2021 04:48:27PM via free access man (Martin, Tolis, Woods & Guyda, 1979; Janowsky, Judd, Huey, Roitman & Parker, 1979). The twice-daily prolactin surge seen during early pregnancy and pseudopregnancy in rats (Butcher, Fugo & Collins, 1972) and mice (Barkley, Bradford & Geschwind, 1978) has not been studied in relation to the opiates. This pattern of prolactin surges is necessary for maintenance of the corpus luteum during early gestation (Mednick, Bradford & Geschwind, 1980). Blockade of prolactin release by dopamine agonists, such as ergot alkaloids, during this critical period, interrupts pseudopregnancy or pregnancy, with the animals becoming oestrous within 2 days (Shelesnyak, 1955; Carlsen, Zeilmaker & Shelesnyak, 1961). Since endorphins appear to have an important role in many physiological responses involving prolactin, the administration of opiate antagonists during early pregnancy or pseudopregnancy may be expected to block the prolactin surges, and an interruption of the pregnant state, as seen with ergot alkaloids, should result. This possibility was investigated in this study. Materials and Methods Random-bred albino mice, CD-I (Charles Rivers Breeding Laboratories, Wilmington, Massachusetts), were kept on a 14-h light cycle (lights on from 05 :00 to 19:00 h) with constant temperature (22 ± 1°C) and humidity (55%). Food (Wayne Lab Blocks for Rodents) and water were always available. Naltrexone was donated by Endo Laboratories, Inc. (Garden City, New York). Chlornaltrexamine was provided by Dr A. E. Takemori. Bromocriptine (CB-154) was donated by Sandoz Pharmaceuticals (East Hanover, New Jersey). Morphine sulphate was donated by Mallinckrodt, Inc. (St Louis, Missouri). Phenyl/j-quinone was obtained from Sigma Chemical Co. (St Louis, Missouri). Ethanol, reagent grade, was obtained from U.S. Industrial Chemicals Co. (New York, New York). Pseudopregnancy. The naltrexone derivative chlornaltrexamine is an alkylating agent shown to have opiate antagonist activity for at least 72 h after intracerebroventricular (i.c.v.) administration (Portoghese, Larson, Jiang, Takemori & Caruso, 1978). A dosing regimen was used which would provide continuous antagonism before mating and through the first 5 days of pseudopregnancy. Chlornaltrexamine, 1 µg in 10 µ saline (9 g NaCl/1) or saline alone, was injected into females i.c.v. at 16:00 h before the animals were paired with vasectomized males. A second dose of chlornaltrexamine, 1 µg i.c.v., was administered 3 days later, thereby providing opiate antagonism through Day 6 of pseudopregnancy. Mating was confirmed by the presence of a vaginal plug, and this was considered to be Day 1 of pseudopregnancy. The length of pseudopregnancy was defined as the number of days from mating until the occurrence of the next pro-oestrus, as determined by the cell pattern in the vaginal smear (Allen, 1922). In another group of mice, naltrexone, 5 mg/kg, was administered i.p. at 22:00 h on the evening following a pro-oestrous smear. Treated animals and saline-injected controls were mated to vasectomized males that night, and the length of the resulting pseudopregnancy was determined. Pregnancy. The effects of systemic naltrexone and bromocriptine on the maintenance of pregnancy were studied. Treatment groups consisted of: 0-5 mg bromocriptine/mouse s.c, given at 12:00 h on Day 2 of pregnancy; 0-5 mg bromocriptine/mouse s.c. given at 12:00 h on Day 3; naltrexone at 10, 100 or 200 mg/kg given on Day 2; naltrexone at 10 or 100 mg/kg given on Day 3; naltrexone, 3 doses of 100 mg/kg given at 4-h intervals beginning at 09 :00 h on Day 2; 0-2 ml vehicle (20% ethanol in saline) on Day 2. Another experiment examined the effect of naltrexone, 10 or 100 ug in 10 µ saline, given i.c.v. at 12:00 h on Day 2, compared to saline controls. In all these experiments, animals were killed on Days 10-12 and examined for implantation sites. Continuous administration of naltrexone in pregnant mice was achieved by the use of mini-osmotic pumps (Alza Corp., Palo Alto, California). Model 2001 minipumps with an Downloaded from Bioscientifica.com at 10/03/2021 04:48:27PM via free access estimated in-vivo delivery rate of 0-9 µ /h were filled with a naltrexone solution to give an estimated delivery rate of 12-3 µg/h (10 mg/kg/day in a 30-g mouse). Minipumps were implanted subcutaneously, under light ether anesthesia, at 12:00 h on Day 1 of pregnancy. Animals were killed on Day 9 and examined for implanted embryos. The maintenance of effective levels of antagonist was tested on the morning of Day 9. The ability of 10 mg morphine sulphate/kg to provide analgesia in the phenylquinone writhing test (Blumberg, Wolf & Dayton, 1965) was determined in mice with implanted naltrexone or saline-filled minipumps. Failure of this dose of morphine (which was 10 times the ED50 in this population) to provide analgesia was considered proof of significant antagonism. In cases where morphine did produce analgesia, reversal with 1 mg naltrexone/kg i.p. was examined. Results Pseudopregnancy. The effects of opiate antagonists on duration of pseudopregnancy is shown in Table 1. Chlornaltrexamine, given both before mating and 3 days after, had no effect on the length of pseudopregnancy. Likewise, naltrexone administered before mating did not affect the initiation of pseudopregnancy or its duration. Table 1. Effect of opiate antagonists on the length of pseudopregnancy in mice No. of Mean ± s.e.m. duration Treatment mice of pseudopregnancy (days) Saline*, 2 10 µ i.c.v. 9 9-0 ±0-2 Chlornaltrexamine*, 2 1 µg/mouse in 10 µ 11 8-7 + 0-3 Salinet.0-2mli.p. 9 10-4 ±0-9 Naltrexonet, 5 mg/kg i.p. in 0-2 ml 10 110 ± 1-3 * Chlornaltrexamine or saline was given immediately before pairing with males (16:00 h). A second dose was given 3 days later. t Naltrexone or saline was given at 22 :00 h on the night after a pro-oestrous vaginal smear. Table 2. Effects of bromocriptine and naltrexone on early pregnancy in mice Treatment No. of mice No. of implantations/ Pregnant on pregnant mouse Drug Dose Route Dayt Treated Days 10-12 (mean ± s.e.m.) Vehicle* s.c. 15 15 •3 + 0 Bromocriptine 0-5 mg/mouse S.C. 11 8 5 ±0 10 2 •0±4 Naltrexone 10 mg/kg 5 4 5±0 11 9 •9 + 0 100 mg/kg 5 5 •2± 1 6 6 •7± 1 200 mg/kg S.C. 8 7 •1 ±0 3 100 mg/kgt S.C. 6 5 2± 1 Saline 10 µ] i.c.v. 6 4 •0± 1 Naltrexone 10 µ i.c.v. 5 •2±0 100 µ8 i.c.v. 7 •0 + 0 * Because of the solubility characteristics of bromocriptine, all s.c.