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. injections were given in 0-2 ml of 20% ethanol in saline. t Three injections of 100 mg/kg at 4-h intervals beginning at 09 :00 h. Presence of a vaginal plug was Day 1 of pregnancy (approximately 12 h after mating).

Downloaded from Bioscientifica.com at 10/03/2021 04:48:27PM via free access Pregnancy. Bromocriptine and various doses of naltrexone were given to pregnant mice on Days 2 or 3 (Table 2). Bromocriptine administered on Day 2 had only minimal effects, interrupting pregnancy in only 3 of 11 mice. Administration on Day 3 was much more effective with 1 of the 2 mice still pregnant having only 4 implantations. Naltrexone given on either Days 2 or 3, all 4 doses, had no consistent effect on the establishment of pregnancy. In all mice remaining pregnant, a normal number of implantations (8-14) was observed. Multiple dosing on Day 2 did not increase the effectiveness of naltrexone. Doses of 100 or 200 mg/kg produced neurological effects, including mild tremors and catatonia in many mice. Administration of naltrexone i.c.v. did not produce an effect in mice when compared to the saline-treated controls; the latter had a lower pregnancy rate than did the s.c. saline control group. The effectiveness of the minipumps in maintaining antagonistic levels of naltrexone in 5 chronically treated mice was demonstrated by the failure of 10 mg morphine/kg to provide analgesia. In the 5 saline-treated mice (0-9 µ /h/mouse), 10 mg morphine sulphate/kg prevented writhing in response to 2 mg phenylquinone/kg. In these animals, analgesia was blocked by 1 mg naltrexone/kg i.p. Four out of five of the saline-treated and all 5 of the naltrexone-treated mice were pregnant on Day 9, with no difference in the number of implantations present (12-7 ± 1-4 and 11-8 ± 0-7, respectively).

Discussion

Opiate antagonists administered before mating to sterile males had no effect on the length of the resulting pseudopregnancy. Guidotti & Grandison (1979) have shown that 1 mg naltrexone/kg lowers basal prolactin levels in male rats for 3-4 h. The 5 mg/kg dose given in this study would be expected to provide substantial antagonism during a period encompassing the presumed time of mating (Snell, 1941). If an opiate receptor system is involved in the prolactin release seen after cervical stimulation during mating (Sinha, Wickes & Baxter, 1978), it should be blocked by this treatment. The failure of naltrexone to prevent or shorten pseudopregnancy can be taken as evidence that the initial stimulation of the neural components responsible for semicircadian prolactin release is not dependent on endorphin activity. The long-acting antagonist chlornaltrexamine also had no effect on the length of pseudopregnancy although antagonism was maintained from well before mating until Day 6 of pseudopregnancy. These results further substantiate the findings with naltrexone and also indicate that an endorphin input is not necessary for the continued maintenance of pseudopregnancy. Bromocriptine and various dosing regimens of naltrexone were tested for their effects on maintenance of pregnancy. Previous studies in mice have demonstrated a limited time period in which ergot alkaloids can disrupt pregnancy. Milligan, Khan & Thorne (1980) found bromocriptine to be effective throughout the first 6 days of pregnancy, similar to the pattern seen in rats (Shelesnyak, 1955). Mantle (1969) reported that argoclavine (an alkaloid similar to ergots) was effective only on Days 3-6, while Carlsen et al (1961) could interrupt pregnancy with ergocornine only on Days 4 and 5. The results of the present study are consistent with a limited effective period starting on Day 3. In view of these results, the effects of naltrexone administered on Days 2 or 3 were tested. At doses ranging from 10 to 200 mg/kg s.c, minimal effects were seen. Pregnancy was prevented in up to 20% of animals within some treatment groups, but this effect was not dose dependent. Even three consecutive doses of 100 mg/kg did not result in any consistent effect. Choudary & Greenwald (1969), in a study of a large group of Swiss albino mice, found that 12-8% of females with vaginal plugs were not pregnant on Days 6-11. In view of this, the effects seen in the naltrexone-treated mice probably are not significant despite the 100% pregnancy rate observed in the control group. In all cases, the number of implantations in pregnant animals was not different from controls or from that reported

Downloaded from Bioscientifica.com at 10/03/2021 04:48:27PM via free access previously for similar mice (Choudary & Greenwald, 1969). Naltrexone i.c.v. also had little effect when compared to saline controls, both having pregnancy rates between 60 and 80%. Constant administration of naltrexone from 12:00 h on Day 1 of pregnancy had no effect on the maintenance of pregnancy or the number of implantations. Morphine antagonism was demonstrated just before death on the 8th day after minipump implantation, which is the end of its expected life-span. Presumably, since antagonism was shown at that time, significant levels of naltrexone were maintained throughout the experiment. These results indicate that, as in pseudopregnancy, an endorphin input may not be necessary for prolactin release. The failure of these opiate antagonists to affect pregnancy or pseudopregnancy consistently is notable in view of the profound effect these agents have on prolactin release in other models. Although prolactin levels were not measured in this study, the lack of effect of these treatments indicates that prolactin release was not affected to the degree seen in mice treated with ergot alkaloids.

We thank Daniel Hendrix for his excellent technical assistance in this study, and the expert consultations from Dr . P. Plotnikoff.

References

Allen, E. (1922) The oestrous cycle in the mouse. Am. Guidotti, . & Grandison, L. (1979) Participation of J. Anat. 30,297-371. endorphins in the regulation of pituitary function. In Barkley, M.S., Bradford, G.E. & Geschwind, I.I. (1978) Endorphins in Mental Health Research, pp. 416- The pattern of plasma prolactin concentration during 422. Eds W. E. Bunney & N. S. Kline. Oxford the first half of mouse gestation. Biol. Reprod. 19, University Press. New York. 291-296. leiri, T., Chen, H.T., Campbell, G.A. & Meites, J. (1980) Blumberg, H., Wolf, P.S. & Dayton, H.B. (1965) Use of Effects of naloxone and morphine on the proestrous writhing test for evaluating analgesic activity of surge of prolactin and gonadotropins in the rat. narcotic antagonists. Proc. Soc. exp. Bio!. Med. 118, Endocrinology 106, 1568-1570. 763-766. Janowsky, D.. Judd, L., Huey, L., Roitman, N. & Bruni, J.F., Van Vugt, D., Marshall, S. & Meites, J. Parker, D. (1979) Naloxone effects on serum growth (1977) Effects of naloxone, morphine, and methion- hormone and prolactin in man. Psychopharma- ine enkephalin on serum prolactin, luteinizing hor¬ cology 65, 95-97. mone, follicle stimulating hormone, thyroid stimulat¬ Mantle, P.G. (1969) Interruption of early pregnancy in ing hormone and growth hormone. Life Sci. 21, mice by oral administration of agroclavin and 461-466. sclerotia of Claviceps fusiformis (Loveless). J. Butcher, R.L., Fugo, N.W. & Collins, W.E. (1972) Reprod. Fert. 18,81-88. Semicircadian rhythm in plasma levels of prolactin Martin, J.B., Tolis, G., Woods, I. & Guyda, H. (1979) during early gestation in the rat. Endocrinology 90, Failure of naloxone to influence physiological growth 1125-1127. hormone and prolactin secretion. Brain Res. 168, Carlsen, R.A., Zeilmaker, G.H. & Shelesnyak, M.C. 210-215. (1961) Termination of early (pre-nidation) pregnancy Mednick, D.L., Barkley, M.S. & Geschwind, I.I. (1980) in the mouse by single injection of ergocornine Regulation of progesterone secretion by LH and methanesulphonate. J. Reprod. Fert. 2, 369-373. prolactin during the first half of pregnancy in the Choudary, J.B. & Greenwald, G.S. (1969) Ovarian mouse. J. Reprod. Fert. 60, 201-207. activity in the intact or hypophysectomized pregnant Meites, J., Bruni, J.F., Van Vugt, D.A. & Smith, A.F. mouse. Anat. Ree. 163, 359-372. (1979) Minireview: relation of endogenous opioid Deyo, S.N., Swift, R.M. & Miller, R.J. (1979) Morphine peptides and morphine to neuroendocrine functions. and endorphins modulate dopamine turnover in rat Life Sci. 24, 1325-1336. median eminence. Proc. natn. Acad. Sci. U.S.A. 76, Milligan, S.R., Khan, A.A. & Thorne, B.C. (1980) 3006-3009. Delayed pseudopregnancy in the mouse. J. Reprod. Ferland, L., Kledzik, G.S., Cusan, L. & Labrie, F. (1978) Fert. 60,49-51. Evidence for a role of endorphins in stress- and Muraki, T., Nakadate, H.. Tokunaga, Y., Kato, R. & suckling-induced prolactin release in the rat. Molec. Makino, T. (1979) Effect of narcotic analgesics and cell. Endocr. 12, 267-272. naloxone on proestrus surges of LH, FSH, and Grandison, L. & Guidotti, A. (1977) Regulation of prolactin in rats. Neuroendocrinology 28, 241-247. prolactin release by endogenous opiates. Nature Portoghese, P.S., Larson, D.L., Jiang, J.B., Takemori, Lond. 270, 357-359. A.E. & Caruso, T.P. (1978) 6B-(N,N-Bis (2-chloro-

Downloaded from Bioscientifica.com at 10/03/2021 04:48:27PM via free access ethyl)am¡no-17-(cycloprophyl-methyl)-4,5a-epoxy- Laboratory Mouse, pp. 55-88. Ed. G. D. Snell. 3-14 dihydroxymorphinan (Chlornaltrexamine). a McGraw-Hill, Philadelphia. potent opioid receptor alkylating agent with ultra- Takehara, J., Kageyama, J., Yunoki, S.. Yakushiji, W., long narcotic antagonist activity. J. med. Chem. 21, Yamauchi, J., Kagehama, N. & Ofuji, T. (1978) 598-599. Effects of 2-Bromo-a-ergocryptine on ß-endorphin- Rivier, C, Vale, W., Ling, N., Brown, M. & Guillemin, induced growth hormone, prolactin and luteinizing R. (1977) Stimulation in vivo of the secretion of hormone release in urethane anesthetized rats. Life prolactin and growth hormone by ß-endorphin. Sci. 22, 2205-2208. Endocrinology 100, 238-241. Van Vugt, D.A., Bruni, J.F. & Meites, J. (1978) Shelesnyak, M.C. (1955) Disturbance of hormone Naloxone inhibition of stress-induced increase in balance in the female rat by a single injection of prolactin secretion. Life Sci. 22, 85-90. ergotoxine ethanesulphonate. Am. J. Phvsiol. 180. Van Vugt, D.A., Bruni, J.F., Sylvester, P.W., Chen, H.T., 47-49. Ieiri, T. & Meites, J. (1979) Interaction between Sinha, Y.N., Wickes, M.A. & Baxter, S.R. (1978) opiates and hypothalamic dopamine and prolactin Prolactin and growth hormone secretion and mam¬ release. Life Sci. 24, 2361-2367. mary gland growth during pseudopregnancy in the mouse. J. Endocr. 77, 203-212. Snell, G.D. (1941) Reproduction. In Biology of the Received 13 August 1981

Downloaded from Bioscientifica.com at 10/03/2021 04:48:27PM via free access