Opioidergic Control of Luteinizing Hormone Release in the Female Rabbit: Influence of Ovariectomy and Steroid Replacement on Pulsatile Secretion

Transcription

1 BIOLOGY OF REPRODUCTION 39, (1988) Opioidergic Control of Luteinizing Hormone Release in the Female Rabbit: Influence of Ovariectomy and Steroid Replacement on Pulsatile Secretion E. V. YOUNGLAI,2 3 N. THOMPSON,3 and M. WILKINSON4 Department of Obstetrics & Gynecology3 McMaster University Health Science Centre Hamilton, Canada Ontario L8N3Z5 and Department of Physiology & Biophysics Dalhousie University Halifax, Nova Scotia Canada B3H 4H7 ABSTRACT The influence of ovariectomy and steroid replacement on naloxone-induced changes in pulsatile secretion of luteinizing hormone (LH) in the female rabbit was examined. Blood samples were taken every 5 mm through an indwelling catheter in the rabbit ear artery, and plasma was stored until assayed for LH by established radioimmunoassay procedures. In the intact animal, saline injection had no effect on LHsecretion. Although naloxone (10 mg/kg) caused a 7-fold increase in mean LH pulse amplitude by 30 mm after injection, this increase was not statistically significant because 5 of 11 animals did not respond. In animals ovariectomized 48 h previously, naloxone significantly increased LH concentration by 194% at 23 mm after injection. When long-term ovariectomized rabbits were treated with estradiol benzoate and then were given naloxone, no significant increase in LH was observed, although many animals did respond. Treatment of long-term ovariectomized rabbits with 1 j.zg estradiol benzoate and 100 pg progesterone or 1 mg testosterone propionate on Days I and 3 and naloxone on Day 4 resulted in a significant increase in LH mm later. Although there was an increase in pulse amplitude, no change was detected in pulse frequency after naloxone. These data suggest that the hypothesis of steroidopioid coupling in the control of LH secretion is not applicable to the female rabbit. INTRODUCTION Studies from our laboratory have shown that there is an age-dependent change in the binding of the opiate antagonist naloxone during development in the female rabbit (Wilkinson and YoungLai, 1986). The changes in naloxone binding have some relationship to changing patterns of gonadotropin secretion. In further studies it was observed that the sensitivity of the hypothalamus to naloxone changed according to the age of the rabbits (YoungLai et a!., 1988b). In view of the known phenomenon of pulsatile hormone release in a number of animal species, including man Canada. Accepted May 23, Received February 19, This work was supported by the Medical Research Council of 2 Reprint requests. (Dierschke et al., 1970; Foresta et al., 1983; Kalra and Kalra, 1983; Barraclough et a!., 1984; Ferin et a!., 1984; Brooks et al., 1986), it was decided to reexamine the role of endorphins in regulating pu!satile gonadotropin release in the female rabbit. The ovariectomized rabbit was used to amplify the pu!satile luteinizing hormone (LH) release patterns. MATERIALS AND METHODS Female New Zealand white rabbits approximately 3 kg in weight were obtained from local breeders. They were housed under controlled lighting (lights on h) with free access to food and water. They were acclimatized to these laboratory conditions for at least 1 wk before use. At the time of experimentation, animals weighed kg. Ovariectomies were aseptically performed through a midline ventral incision under sodium pentobarbital 630

2 OPIOID CONTROL OF RABBIT LH RELEASE 631 anesthesia. Animals were allowed to recover for 48 h before experimentation. The following groups of animals were used: a) normal intact adult females. b) normal intact adult females treated with estradio! benzoate on Days 1 and 3 and injected with naloxone or saline on Day 4. c) acutely ovariectomized, i.e. 48 h post-surgery and given saline or na!oxone. d) long-term ovariectomized (LTO), i.e. more than 21 days post-surgery, and given saline or naloxone. e) LTO and estradiol benzoate (EB, 1 pg/rabbit), on Days 1 and 3, followed by naloxone on Day 4. f) LTO and EB (1 pg/kg) on Days 1 and 2, followed by naloxone on Day 4. g) LTO and combination EB (1 pg)/progesterone (100 j.zg) on Days 1 and 3, followed by naloxone on Day 4. h) LTO and testosterone propionate (TP, 1 mg), on Days 1 and 3, followed by naloxone on Day 4. Steroids were obtained from Steraloids Inc., Wilton, NH. Naloxone hydrochloride was purchased from Sigma Chemical Co., St. Louis, MO sodium iodide of high specific activity was purchased from NEN, Boston, MA. Steroids were dissolved in sesame oil prior to s.c. injection. Naloxone in saline (10 mg/kg, mg/ml) was then given on Day 4. An indwelling Teflon catheter, 3.2 cm long, 20 gauge (Deseret Pharmaceutical Co., Sandy, UT) was placed in the middle ear artery and taped down for blood sampling. Blood (0.7 ml) was withdrawn every 5 mm for 1 h prior to the injection of naloxone, 10 mg/kg, in saline. Controls received saline. Blood samples were drawn for another 2-3 h. Plasma was stored at -20#{176}C until analyzed for LH by established radioimmunoassay procedures (Moor and YoungLai, 1975). The intra- and interassay coefficients of variation were less than 5% and 18%, respectively. Results are expressed in terms of the pure rabbit LH standard E X 130 GB provided by Dr. H. Papkoff. Reagents for radioimmunoassay of LH were obtained from Dr. A. F. Parlow, Torrance, CA. Estradiol, testosterone and progesterone were measured by radioimmunoassay using specific antisera, except for testosterone, which had significant cross-reaction with dihydrotestosterone (YoungLai, 1985). LH values were subjected to the Pulsar Program analysis of Merriam and Wachter (1982) kindly provided by Dr. J. F. Gitzen, University of Illinois at Urbana-Champaign. For the detection of pulses and mean output, we used a 1% error rate for the calculations of the G values according to a normal distribution probability. The Pulsar program provided data on peak amplitude, peak length, frequency, and interpeak intervals for each animal. In most cases, because of the duration of sampling after naloxone injection, peak lengths, frequency, and interpeak interval were not analyzed further. The mean pulse amplitude was compared before and after naloxone treatment by the paired t-test. Preinjection mean values were also compared between treatment groups using the unpaired t-test. Another type of analysis was carried out to determine the peak height when the observed peak response was not symmetrical. In these cases, multiple linear regression was carried out on at least 5 points around an apparent peak and the predicted peak was calculated from the computer program. These derived peak values were compared against the mean prenaloxone LH values to determine whether a response occurred. Analysis was carried out with the Mann- Whitney U test for nonparametric data. A number of intact females were killed the day following naloxone injections and their hypothalami were removed for determination of opioid receptors as previously described (Wilkinson and YoungLai, 1986). RESULTS The secretion of LH in the normal intact adult rabbit was found to be pu!sati!e with a frequency of Although levels were generally about 1 ng/ml, there was an occasional female who would show pulses greater than 4 ng/m! (Fig. la). Saline injections had no effect on pulse amplitude whereas naloxone at 10 mg/kg caused an increase in LH secretion that, in some instances, mimicked an ovulatory LH surge (Fig. ib). A total of 7 animals were used as controls with saline given 1 or 2 h after the start of blood sampling. The pulse amplitude was 0.37 ng/m! with peak length of 16.6 mm, frequency of , and interpeak interval of 30.5 mm. After naloxone injection, peak length generally increased, and although the pulse amplitude increased from 0.47 ± 0.14 ng/ml to 3.38 ± 2.01 ng/mi after naloxone (Table 1), this increase was not statistically significant. Of the 11 females used to test the effects of naloxone, 5 showed no increase in LH and 6 had a peak in LH

3 632 YOUNGLAI ET AL. when values were subjected to multiple linear regression analysis. The mean time interval between naloxone injection and peak LH value was 41 mi In the 6 females, the LH peak was 2.7- to 18.6-fold I -j TIME - mm FIG. 1. Luteinizing hormone (LH) secretion in adult female rabbits given (a) saline (S) 2 h or, (1,) naloxone (N, 10 mg/kg s.c.) 30 mm after the start of blood sampling. Each line represents one animal. Note the log V-axis in (b). Arrow indicates time of saline or naloxone injection. Five representative animals are shown. greater than the mean levels prior to naloxone injection. One of these rabbits had an LH peak greater than 13 ng/ml. Eight corpora lutea were found in the ovaries of this rabbit the next day. All intact adult rabbits were tested during 2 mo, October and January. There was no difference in estradiol (105 ± 36 pg/ml vs. 78 ± 23 pg/rn!) and testosterone (78 ± 35 pg/ml vs. 73 ± 36 pg/rnl) levels between animals that responded and those that did not. Progesterone levels were 0.82 ± 0.16 ng/ml in the nonresponders and 0.64 ± 0.05 ng/ml in the responders, but the difference was not significantly different because of heterogenous variances. Six of these normal intact rabbits were killed the next day and hypothalarni were taken to determine the maximum binding sites for [3H]na!oxone (Wilkinson and YoungLai, 1986). As shown in Figure 2, there was no obvious difference in hypothalamic binding capacity of rabbits that responded and those that did not. The differences between responders and nonresponders in body weights (2.80 ± 0.04 kg vs ± 0.09 kg) and uterine weights (6.45 ± 0.08 g vs ± 0.17 g) were also not significant. TABLE 1. Changes in pulse amplitude of luteinizing hormone (LW secretion in female rabbits after saline or naloxone injections (mean ± SEM). LH Pulse amplitude Treatment Pre Post p Saline-injected Intact normal (n=7) 0.37 ± ± 0.06 NS Acutely ovex (n=5) 1.08 ± ± 0.28 NS LTO (n=3) 1.97 ± ± 0.10 NS LTO, estrogen-primed (n=4) 0.87 ± ± 0.12 NS Naloxone-injected Intact normal (n=11) 0.47 ± ± 2.01 NS Intact EBbtreated, Days 1, 3 (n=4) 0.29 ± ± 0.43 NS Acutely ovex (n=5) 0.85 ± ± LTO (n=11) 2.00 ± ± LTO + EB, Days 1, 3 (n=7) 1.02 ± ± 1.37 NS LTO + EB, Days 1, 2 (n=5) 1.39 ± ± 0.23 NS LTO + EB/p4c, Days 1, 3 (n=10) LTO + TPd, Days 1, 3 (n=4) 0.81 ± ND 1.63 ± ± alto = long-term ovariectomy. beb = estradiol benzoate. c4 = progesterone. dtp = testosterone propionate. #{149}p, values were determined for mean LH pulse amplitude before and after saline or naloxone with the paired Student s t-test. ND = no pulses were detected in these animals.

4 OPIOID CONTROL OF RABBIT LH RELEASE 633 CI, 10 I- )( E a. C, z z C.) U. C) LU a- C/) FIG. 2. Specific binding of l3hlnaloxone to hypothalamic slices of intact female rabbits the day after naloxone injection. Results are mean ± SEM for replicate slices from the same rabbit. R - Rabbit that showed a significant response to naloxone; NR = nonresponder to naloxone treatment. Figure 3 shows the secretion of LH in female rabbits ovariectomized 48 h previously. As seen in Pane! a, there is a marked increase in pulse amplitude of LH secretion compared to data in Figure la. The effect of naloxone in these animals is shown in Panel b. The peak in LH secretion, which was about 3-fold greater than baseline values, occurred at 23 mm after na!oxone injection. The mean pulse amplitude showed a significant increase from 0.85 ± 0.19 ng/ml to 2.39 ± 0.47 ng/ml (Table 1). All 5 animals used in this experiment showed a peak in LH after naloxone. Figure 4 shows the LH secretion in rabbits ovariectomized at least 5 wk previously and given saline or naloxone. The LH pulse amplitude was 1.97 ± 0.53 ng/m!, which was not significantly different after saline injection, but rose to 3.09 ± 0.69 ng/ml after na!oxone. Al! 11 animals showed a response to naloxone, with a mean interval between injection and peak of 35.5 mm. The range of increases was 1.3- to 14.4-fold. The mean pulse amplitude of these ovariectomized rabbits prior to injection was significantly greater (2.00 ± 0.37 ng/m!) than that of normal intact rabbits (0.37 ± 0.05 ng/ml, Table 1). E 0, C I -J 0, C I -I TIME -mm TIME -mm FIG. 3. Luteinizing hormone (LH) secretion in acutely ovariectomized (2 days post-op) female rabbits given (a) saline, 3 representative animals, or (b) naloxone, 4 representative animals. Symbols are the same as for Figure 1. FIG. 4. Luteinizing hormone (LH) secretion in rabbits ovariectomized for more than 5 weeks and given (a) saline, 3 representative animals, or (b) naloxone, 5 representative animals. Note the log V-axis in (b). Symbols are the same as for Figure 1.

5 634 YOUNGLAI ET AL. When these LTO rabbits were treated with EB (Fig. 5), the mean LH pulse amplitude was reduced to 0.87 ± 0.16 ng/m! and no change was observed after saline injection (Fig. 5a). In animals treated with EB (1 pg/kg) on Days 1 and 2 followed by naloxone on Day 4 (Fig. 5b), the mean LH pulse amplitude rose from 1.39 ± 0.16 ng/m! to 1.67 ± 0.23 ng/ml (Table 1), but 2 of 5 animals did not respond to naloxone. The highest LH level attained was 4.8-fold higher than the mean preinjection level. In this group of animals, naloxone did not increase LH levels. Estradiol levels were 82 ± 22 pg/rn!. In animals treated with EB (1 pg/rabbit) on Days 1 and 3 (Fig. 5c) followed by naloxone on Day 4, the mean LH pulse amplitude rose from 1.02 ± 0.21 ng/ml to 2.84 ± 1.37 ng/m! (Table 1). In these animals, the mean LH pulse amplitude after naloxone was not significantly different from that before na!oxone. However, analysis, by multiple regression, of the LH values within 1 h after na!oxone indicated that a peak of E N a, C E N C -j -J TIME - mm 120 TIME- mm FIG. 5. Luteinizing hormone (LI-I) secretion in rabbits ovariectomized for more than 5 wk pretreated with estradiol benzoate and given (a) saline, 3 representative animals, or (b and c) naloxone. Protocol is the same as for Figure 3. (b) Estradiol benzoate was given on Days I and 2 and naloxone on Day 4, 5 representative animals. (c) Estradiol benzoate was given on Days 1 and 3 and naloxone on Day 4, 5 representative animals. Note the log V-axis in (c). Symbols are the same as Figure 1. FIG. 6. Luteinizing hormone (LH) secretion in rabbits ovariectomized for more than 5 wk and given testosterone propionate or a combination of estradiol benzoate and progesterone on Days 1 and 3 followed by naloxone on Day 4. (a) Saline, 4 representative animals. (b) Naloxone- to estradiol benzoate/progesterone-treated, 5 representative animals. (c) Naloxone- to testosterone propionate-treated (n=4). Saline or naloxone was given after 60 mm of baseline samplings. Symbols are the same as for Figure 1.

6 OPIOID CONTROL OF RABBIT LH RELEASE 635 LH, 2.9-fold higher than the mean values prior to injection, occurred at 25.8 mm after naloxone. Circulating estradio! levels were 59 ± 7 pg/m!. Treatment of LTO rabbits with combination EB (1 pg)/progesterone (100 pg) on Days 1 and 3 depressed mean LH pulse amplitude from 1.97 ± 0.53 ng/ml to 0.81 ± 0.21 ng/ml (Table 1). This mean pulse amplitude increased significantly to 1.63 ± 0.24 ng/m! after naloxone injection (Tab!e 1, Fig. 6b). Of the 10 rabbits used in this experiment, 2 failed to respond to naloxone. The naloxone-induced increases were 1.4- to 3.9-fold greater than preinjection levels. The time interval between injection and peak LH level was 24.5 mm. Circulating estradio! levels were 210 ± 42 pg/rn! and those of progesterone were 1.3 ± 0.1 ng/m!. Similarly, in ovariectomized animals treated with TP (1 mg) on Days 1 and 3, the mean LH pulse amplitude was completely abolished. Na!oxone administration to these animals caused a significant increase in mean LH pulse amplitude to 0.51 ± 0.05 ng/m! (Table 1, Fig. 6c). All 4 rabbits showed a response to naloxone. LH levels prior to na!oxone injection ranged from 0.7 to 1.3 ng/ml but showed no evidence of pu!satility. These levels increased 1.8-fold after naloxone, and the peak levels were found at 19.2 mm after injection. Circulating estradiol levels were 32 ± 6 pg/mi, progesterone, 0.81 ± 0.1 ng/ml, and testosterone, 1130 ± 73 pg/rn!. DISCUSSION In previous communications from this laboratory (YoungLai et a!., 1988a,b), we were unable to detect pulses in gonadotropin secretion when blood samples were taken at 20-mm intervals. The present data, derived from blood samples obtained at 5-mm intervals, indicate that LH is indeed released in a pulsatile manner in intact as we!! as in ovariectomized rabbits and that this pulsatility can be influenced by exogenous steroids. Our results are compatible with recent data (Pau et al., 1986). In the intact female, circulating steroid levels were similar to those reported by other workers (Waterston and Mills, 1976; Lau et al., 1978; Stoufflet and Cai!lo!, 1988). Administration of exogenous estrogen to ovariectomized rabbits resulted in generally lower estradiol levels compared to those in intact animals, except in the combined EB/progesterone group where levels were about 2-fold higher. It is possible that the 100-fold excess progesterone inhibited the clearance of estradiol. The steroid treatment protocol of Paisley and Summerlee (1985) was used to demonstrate a na!oxone-induced increase in LH secretion. Our data suggest that endogenous opioid peptides do have an inhibitory influence on the release of LH, in the intact or the ovariectomized female rabbit, and that steroids are not required to elicit this effect. This confirms a previous report from our laboratory when blood samples were taken less frequently (YoungLai et a!., 1986a,b). In fact, it would appear that the presence of low circulating levels of estradio! may diminish the LH response to naloxone (Table 1) (Orstead and Spies, 1987). Although the lack of response to naloxone has been described under conditions in which gonadal steroid secretion is absent, such as in ovariectomized rats (Bhanot and Wilkinson, 1984; Petrag!ia et a!., 1984) or in postmenopausal women (Reid et a!., 1983; Petraglia et a!., 1986), others have found results similar to ours in ovariectomized ewes (Schi!!o et a!., 1985) and in rats (Fournet et a!., 1985). The reasons for these conflicting results remain unc!ear, although the experiments that have successfully detected na!oxone-induced LH secretion in ovariectomized animals did so with frequent blood sampling (present data; Fournet et al., 1985; Schi!lo et a!., 1985; Orstead and Spies, 1987). In contrast, bolus injections of naloxone followed by a single blood sample did not reveal an increase in LH secretion in castrated rats (e.g. Bhanot and Wilkinson, 1984; Petrag!ia et al., 1984). During preparation of our manuscript, Orstead and Spies (1987) reported that na!oxone can stimulate LH release in the absence of ovarian steroids in the rabbit, which constitutes confirmatory evidence for our data. It has previously been proposed that the negative feedback action of steroids is mediated by opioid peptides, since na!oxone can counteract steroida! effects (Van Vugt et al., 1982; Bhanot and Wilkinson, 1984; Petraglia et al., 1984; Ve!dhuis et al., 1984; Shoupe et a!., 1985). The data obtained in the present studies suggest that endogenous opioids and the negative feedback effects of gonada! steroids are not coupled, but high estradio! levels can effectively prevent the LH-inducing effect of naloxone. Confirmatory evidence for this conclusion has been acquired in the rat (Spencer and Whitehead, 1986) as we!! as the rabbit (Orstead and Spies, 1987). In addition, naloxone has been shown to stimulate gonadotropin-releasing hormone (GnRH) release from

7 636 YOUNGLAI ET AL. the hypothalami of ovariectomized rats (Leadem et al., 1985). Thus, it would appear that in the female rabbit the hypothesis of steroid-opioid coupling in the control of gonadotropin secretion is probably not applicable. The most consistent response to naloxone was observed in the untreated ovariectomized rabbit, especially when given 48 h after surgery (Fig. 3). Although our studies do not indicate the site of action of naloxone on the hypothalamus or pituitary, the data suggest that the acute deprivation of ovarian steroids enhances the opioid inhibition of GnRH release or that the pituitary becomes more sensitive to GnRH. This latter explanation is strengthened by the observation that EB can prevent the naloxoneinduced LH increase (Fig. 5, Table 1) and no significant differences were found in concentrations of progesterone, testosterone, or estradio! between intact females that responded to naloxone and those that did not. It is of interest that Stoufflet and Cai!lol (1988) recently reported that there is no relation between circulating sex steroid concentrations and sexual receptivity in the female rabbit. Progesterone could act at the hypothalamic level by altering the GnRH pulse amplitude and frequency (Ramirez et a!., 1986a; Ramirez and Dluzen, 1986; Lin and Ramirez, 1988). On the other hand, progesterone may counteract the effects of estradiol since combined estradiol/progesterone treatment allows the manifestation of na!oxone-induced LH response (Fig. 6b, Table 1). By contrast, testosterone abolishes LH pulse amplitude in the presence of low levels of estradiol that may have been formed from the exogenous testosterone by peripheral aromatization. We tested the notion that the responsiveness to naloxone in intact rabbits is dependent on hypothalamic binding sites by determining the naloxone binding activity in 6 rabbits after naloxone treatment. In view of the close correlation between loss of opioid inhibition of LH release and decrease in opioid binding in castrated rats (Wilkinson and YoungLai, 1986), we expected to find increased binding in the hypothalamus of the rabbits that responded. No differences were observed (Fig. 2). It could be argued that there may have been a difference at the time of na!oxone injection that would have saturated a!! the binding sites. Because of the rapid reversibility of the opioid binding (Wilkinson and YoungLai, 1986), it may not be possible to obtain more definitive data. In general, our results are similar to data recently published by Orstead and Spies (1987), who used intravenous infusion or intrahypothalamic perfusion. In both studies, na!oxone was effective in stimulating LH release in 50% of intact animals. However, in our studies with intact rabbits, naloxone occasionally induced an LH increase of sufficient magnitude to induce ovulation (Fig. ib, YoungLai et a!., 1988b). 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