Advancement of First Ovulation by the Opioid Antagonist Naltrexone

BIOLOGY OF REPRODUCTION 41, 842-847 (1989) Advancement the Oplold H.M.A. of First Ovulation Antagonist MEIJS-ROELOFS1 Department by Naltrexone and P. KRAMER of Anatomy Faculty Erasnws University 3000DR Rotterdam The Netherlands ABSTRACT The opioid antagonist naltrexone was administered to female rats during the late juvenile period, and its effects on sexual maturation were studied. Naltrexone treatment (2.5 or 20 mg/kg; four daily injections at 2-h intervals) at 28 32 days of age advanced first ovulation in about 55% of the rats. When naltrexone (20 mgI kg) was administered at 30 34 days of age, 75% of the rats responded. In these rats, first ovulation was advanced by 3.4 days and their body weight was 15.1 g lower than in control rats at first ovulation (p<O.Ol). Similar naltrexone treatment at younger (starting on Day 24 or 26) or older (starting on Day 32 or 34) ages did not advance firs: ovulation. The numbers of ova released in advanced, nonadvanced, and control rats were similar. A sign4ficant increase in serum luteinizing hormone (LII) concentration was seen 15 mm after naltrexone injection (20 mg/kg) at all ages studied; the increase was significantly higher (p<O.O5) at 30 days of age than before or after that age. Relatively high response to naltrexone (2.5 mg/kg) was seen from 8 to 4 days before firs: ovulation. Taken together, these data suggest that during the late juvenile stage (8 4 days before firs: ovulation) endogenous peptides critically restrict LII secretion and may constitute a hypothalamic restraint on the onset of puberty. However, changes in pitui:aiy responsiveness to luteinizing hormone-releasing hormone may be part of the mechanism behind the high LII response to naltrexone in rats during the late juvenile stage. - - - INTRODUCTION tion should be sufficient to induce preovulatory changes in the ovaries, whereas sensitivity to naltrexone, reported to decrease near the time of first ovulation (Bhanot and Wilkinson, 1983), would be retained to such a degree that adequate increases in LH secretion could be induced by naltrexone treatment In this study, we tested this assumption. In contrast to earlier reports by Sirinathsinghji et al. (1985) dealing specifically with naloxone, we recently found that neonatal treatment of female rats with opioid antagonists (naloxone or naltrexone) did not specifically influence the timing of sexual maturation (MeijsRoelofs and Kramer, 1988). However, short-term effects of these two opioid antagonists, e.g. increases in luteinizing hormone (LH) secretion, were found; if naltrexone was administered at 28-32 days of age, vaginal opening and first ovulation were clearly advanced in 40% of the rats. From this, we inferred that the influence of this opioid antagonist on pubertal development might be limited to a certain (late juvenile) maturational period. During this period, small increases in LH secre- MATERIALS AND METHODS Female rats of our inbred Wistar substrain (R-Amsterdam) were treated for 5-day periods with four s.c. injections of naltrexone hydrochloride (Sigma Chemical Co., St. Louis, MO) per day (at 1000, 1200, 1400, and 1600 h) at a dose of 2.5 or 20 mg/kg body wt. This treatment schedule was derived from earlier studies on advancement of first ovulation induced by treatment with human choriomc gonadotropin (hCG; Meijs-Roelofs et al., 1985) and appeared to be effective in advancing first ovulation in 40% of the rats treated on Days 28 -32 (Meijs-Roelofs and Kramer, 1988). Accepted July 21, 1989. Received April 7, 1989. tReprmt requests: Dr. H.M.A. Mcijs-Roelofs. Dept. of Anatomy, Medical Faculty. Erasmus University, P.O. Box 1738. 3000DR Rotterdam. The Netherlands. 842 Medical ADVANCEMENT OF FIRST LH and FSH BY NALTREXONE 843 vancement was first ovulation occurring at an earlier age than in control rats (<36 days). Nonadvanced naltrexone-injected rats (n=7) showed first ovulation at an age (37.6 ± 0.6 days) and body weight (86.3 ± 3.2 g) not different from control rats; in advanced rats (n=9), both age and body weight were significantly (p.<O.O1) lower than in control rats. Naltrexone injections at a dose of 20 mg/kg body wt begun at 28 days of age similarly advanced first ovulation in 10 of 18 rats; first ovulation in these advanced rats occurred at a significantly (p<O.O1) lower age and body wt than in control rats or in nonadvanced naltrexone-treated rats (Table 2). When the same treatment schedule was started at 30 days of age, first ovulation was significantly advanced in 7 of 9 rats. Injections of 20 mg/kg body wt begun earlier (Day 24 or Day 26) or later (Day 32 or Day 34) did not advance first ovulation in any of the naltrexone-treated rats. Numbers of tubal ova/corpora lutea found in naltrexone-treated, advanced (10.8 ± 0.3, n=17) and naltrexone-treated, nonadvanced rats (10.6 ± 0.2, n=53) were not different and were comparable to those in salinetreated rats (10.6 ± 0.2, n=63). At all ages studied, serum LH concentration in naltrexone-treated rats increased significantly (p<O.Ol), Assay The concentrations of LH and FSH in serum were estimated by radioimmunoassay (Meijs-Roelofs and Kramer, 1988) and expressed as jtg reference preparation per liter (NIADDK-rat-LH or FSH RP-l). Statistical I Analysis -J Results were analyzed by ANOVA and Duncan’s multiple range test. The Wilcoxon-Mann-Whitney rank sum test was used to compare age and body weight at first ovulation in nalirexone-treated and in control rats. A difference was considered to be significant if the double-tail probability was <0.05. .4- 0 0 0 RESULTS Naltrexone at a dose of 2.5 mg/kg body wt did not advance first ovulation if injections were started on Day 24 or Day 26 (Table 1). However, injections started on Day 28 resulted in occurrence of first estrus in naltrexone-treated rats at a significantly lower body weight than in control rats (83.8 vs. 91.1 g, Table 1). Naltrexone-treated rats were then subdivided into groups designated “advanced” and “nonadvanced.” Criterion for ad- 0 24 26 28 30 Age (days) 32 34 FIG. 1. Serum LH concentrations (mean ± SEM) in 24- to 34-day-old female rats at 15 mm after first injection of 20mg nallrexonedkg body wt (striped bars) or saline (open bars). There were 7-10 rats per group; the difference between naluexone-injected and saline-injected rats was significant (p.d).Ol) at all ages. Injections were started at 24, 26, 28, 30, 32, or 34 days of age. Control rats were injected with 0.1 ml 0.9% NaC1. Rats were checked daily for vaginal opening, and from then on, daily vaginal smears were taken. On the day of first ovulation (estrous smear), rats were killed, tubal ova were counted, the ovaries were removed and prepared for histological examination, and corpora lutea were counted. Rats were bled for LH determinations 15 miii after the first naltrexone (20 mg/kg) or saline injection on the first day of treatment (Days 24, 26, 28, 30, 32, and 34) b (...truncated)