Luteinizing hormone secretion following intracerebroventricular administration of morphine in the prepuberal gilt

Life Sciences, Vol. 45, pp. 691-696 Printed in the U.S.A.

Maxwell Pergamon Macmillan

LUTEINIZING HORMONE SECRETION FOLLOWING INTRACEREBROVENTRICULAR ADMINISTRATION OF MORPHINE IN THE PREPUBERAL GILT C. Richard Barb*', Rhonda D. Kineman**2, James S. Kesner*3 , George B. Rampacek**, and Robert R. Kraeling* USDA-ARS, Richard B. Russell Agricultural Research Center, Athens, GA 30613 and **Animal and Dairy Science Department, University of Georgia, Athens, GA 30602

(Received in final form June 16, 1989)

Antagonism of endogenous opioids with naloxone stimulates luteinizing hormone (LH) release in mature but not prepuberal gilts. The present report demonstrates that the opiate agonist morphine (500 pg), administered intracerebroventricularly (ICV), reduced LH secretion in both ovariectomized mature and prepuberal gilts. We suggest that opioid receptors are functionally coupled to the GnRH secretory system in prepuberal gilts even though endogenous opioid peptide modulation of LH secretion was not demonstrable in our previous studies. Onset of puberty in gilts and females of other species is accompanied by accelerated luteinizing hormone (LH) pulse frequency and decreased hypothalamic sensitivity to the negative feedback action of ovarian steroids (1, 2, 3, 4). These neuroendocrine events represent maturational changes of mechanisms within the central nervous system (CNS) that may be dependent as well as independent of ovarian steroid feedback (2,5). Endogenous opioid peptides (EOP) are involved in neuroendocrine control of LH secretion (6, 7, 8). Several reports suggest that hypothalamic EOP mediate gonadal steroid negative feedback in This laboratory reported previously that prepuberal female rats (9, 10, 11). antagonism of EOP activity with naloxone increased LH secretion in mature gilts given progesterone, but failed to alter LH secretion in prepuberal gilts given progesterone (12). This result may reflect the absence of a functional link between the EOP neurons and the gonadotropin releasing hormone (GnRH) neurons in the prepuberal gilt or absence of EOP receptors functionally coupled to the GnRH secretory system. 'To whom correspondence should be addressed. Department of Anatomy & Cell Biology, Medical 2Present Address: University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425 'Present Address: NIOSH-Experimental Toxicology Branch, Robert A. Taft Laboratories, 4676 Columbia Parkway, Mail Stop C-23, Cincinnati, Ohio 45226

0024-3205/89 $3.00 +.OO

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LH After Morphine in Prepuberal Gilts

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To address the later possibility, the EOP agonist morphine was administered intracerebroventricularly (ICV) to prepuberal gilts and changes in circulating LH concentrations monitored.

Six prepuberal gilts, 130 days of age, and 5 mature gilts, which had displayed one or more estrous cycles of 18-22 days, were ovariectomized (OVX). At the time of OVX, gilts were classified as prepuberal if their ovaries were devoid of corpora lutea and corpora albicantia. The average age of puberty in the population of gilts from which these animals were obtained was 201 t 9 days. Following OVX, animals were individually penned in an environmentally controlled building until the end of the study. Between the second and fourth week after OVX, each prepuberal and mature gilt was surgically implanted with a lateral ICV cannula using the stereotaxic procedure of Estienne et al. (13). All animals were fitted with an indwelling jugular vein cannulae (14) 15 f 2 days after the last ICV surgery and 24 h before treatment. Gilts were randomly assigned to a 2 x 2 factorial arrangement of treatments with main effects of age (prepuberal vs mature) and treatment (morphine vs vehicle). At the time of treatment, mature and prepuberal gilts weighed 113.5 + 2.9 kg and 80.0 + 1.4 kg, and were 260 and 167 days of age, respectively. Blood samples were collected from the jugular vein cannula every 15 min for 3 h before and 3 h after ICV injection of either 500 rg morphine (Eli Lilly Co., Indianapolis, IN) in 250 ~1 of artificial cerebrospinal fluid (CSF; 15) or 250 ~1 CSF vehicle. The experiment was repeated six days later with treatments reassigned in a crossover arrangement within age group. Blood samples were stored for 24 h at 4'C and serum harvested after centrifugation and stored at -2O'C. Serum LH was quantified by using a heterologous radioimnunoassay (14). The reference standard was USDA-pLH-Bl. Sensitivity of the assay was 0.15 ng/ml of serum. Intraassay and interassay coefficients of variation were 8.4% and 10.5%, respectively. To determine the effect of morphine treatment on LH secretion, data were subjected to a general linear model split plot-in-time analysis of variance procedure of the Statistical Analysis System (SAS; 16). The data were analyzed with age, pig, and period as discrete (class) variables. Age and period and the period X age interaction were tested with pig-within-age and period X pig-within-age as the error terms, respectively. Sampling time was divided into five periods. Period One represented the mean of samples collected prior to morphine treatment. The remainder of the sampling time was divided into four l-h periods. Differences between period means within age groups were determined by least-squares contrasts (16). To calculate within animal basal and pulse characteristics for serum LH profiles the following procedure was used. First, overall mean serum LH concentrations were calculated for each animal. Second, one or more consecutive samples in which serum LH concentrations were greater than one standard deviation above the overall mean comprised an LH pulse (12). Basal serum LH concentration for each gilt was the mean of the samples that did not comprise a pulse. Pulse amplitude was the highest value above basal minus the basal value. To evaluate the effect of treatment between ages on overall mean serum LH concentrations, number of LH peaks, and amplitude of LH peaks, the difference between the before and after treatment means were subjected to analysis of variance using the general linear model procedures of SAS (16). In addition, to evaluate the effect of treatment within age on the above mentioned LH parameters the before and after treatment means were subjected to analysis of variance procedure of SAS (16). Differences between treatment means were determined by least-squares contrasts (16).

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Results Mean serum LH concentrations, as well as serum LH pulse frequency and LH pulse amplitude were unaffected by CSF injection and averagea 0.96 f 0.07 ng/ml, 2.56 f 0.24/3 hour and 0.43 f 0.05 ng/ml for mature gilts and 1.02 f 0.04 ng/ml, 2.83 f 0.17/3 hour and 0.67 f 0.03 ng/ml for prepuberal gilts, respectively, throughout the sampling period. Pretreatment serum LH concentrations were greater (Pt0.05) in prepuberal compared to mature gilts receiving morphine. Morphine decreased (Pt0.05) serum LH concentration and LH pulse frequency in prepuberal and mature gilts, while LH pulse amplitude was unaffected by morphine treatment (Table 1).

TABLE I Serum LH Concentrations, LH Pulse Frequency and LH Pulse Amplitude in Ovariectomized Mature and Prepuberal Gilts Before and After Intracerebroventricular Injection of Morphine.a

Item

Mature

Prepuberal Morphine

Before

After

Before

After

Serum LHbgc,d concentrations (ng/ml)

0.73 f 0.10

0.45 f 0.13

1.16 f 0.08

0.60 f 0.06

LH pulsed frequency (pulses/3 h)

2.80 f 0.40

1.40 f 0.70

3.00 f 0.30

0.70 f 0.30

LH pulsee amplitude

0.36 f 0.05

0.40 f 0.10

0.69 i 0.08

1.04 f 0.22

(w/ml 1 aMean f SEM bBefore treatment means differ (Pt0.05). 'Magnitude of change after treatment differs between ages (PcO.05). dBefore and after treatment means differ (PcO.05). eBefore and after treatment means differ between ages (PxO.05). Morphine treatment resulted in an acute and rapid decrease (Pt0.05) in LH secretion in the prepuberal gilts and averaged 0.86 f 0.09 ng/ml 'during the first h. Serum LH concentrations continued to decrease (Pt0.05) and averaged 0.42 f 0.09 ng/ml during the second h after morphine and remain suppressed for the remainder of the sampling period. However, in mature gilts serum LH concentrations did not change until the second h after morphine and averaged 0.46 f O.lOng/ml and were suppressed for the rest of the treatment period (Fig. 1).

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LH After Morphine in Prepuberal Gilts

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o--o 0-0

0.0

’ i

-180

I

I

-120

-60

I

I

0

60

Mature Prepuberal

I

120

I

180

Minutes FIG. 1 Serum LH concentrations (x f SEH) before and after morphine treatment (treatment = Time 0) in ovariectomized mature and prepuberal gilts. piscussion Evidence suggests that EOP are involved in mechanisms that suppress LH secretion in the pig: proopiomelanocortin (POMC) containing neurons are located in the vicinity of the GnRH neurons in the hypothalamus (17, 18) and naloxone, an opioid antagonist, increased LH secretion in the sexually mature pig (19, 20). Previous work from this laboratory demonstrated that doses of 1,3 or 6 mg/kg body weight (EN) of naloxone failed to alter LH secretion in intact prepuberal gilts. In addition, serum LH concentrations were unchanged after naloxone (lmg/kg 8W) in OVX, progesterone-treated prepuberal gilts. However, LH secretion increased when gilts were OVX prepuberally and treated with progesterone and naloxone at a chronological age when puberty occurred in intact contemporary gilts (12). We concluded that development of EOP modulation of LH secretion in the prepuberal gilt is a brain maturational process. The present results indicate, however, that EOP receptors are anatomically linked to the GnRH/LH secretory system in the prepuberal gilt. These results along with our previous work (12) are similar to results from work in the infantile male rat, in which LH secretion was decreased by morphine while naloxone was ineffective in altering serum LH concentrations (21). The authors concluded that EOP neurons are not functionally coupled to the GnRH system at that stage of development. The ability of morphine to decrease LH pulse frequency in both prepuberal and mature gilts suggests that the frequency of hypothalamic discharge of GnRH was inhibited. An abundance of evidence indicates that EOP inhibition of LH secretion is by a hypothalamic site of action (13, 22, 23, 24). We previously demonstrated that 500 pg morphine ICV inhibited pulsatile secretion of LH compared to lower doses of morphine (13). Armstrong et al.

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(25) reported that morphine prevented the increase in LH secretion associated with transient weaning in the postpartum sow, whereas Trudeau et al. (26) demonstrated that morphine was ineffective in altering LH secretion in intact or castrated 6 week old male pigs. However, in the studies of Armstrong et al. (25) and Trudeau et al. (26) morphine was administered intravenously and cannot be compared with ICV administration. In the present study, morphine treatment resulted in an acute and rapid decrease in LH secretion in prepuberal gilts compared to mature gilts which is consistent with an age related reduction in LH sensitivity to morphine. Bhanot and Wilkinson (9) demonstrated that LH sensitivity to FK33-824 (Met-enkephalin agonist) decreased with age in the prepuberal female rat. They suggested this may be the basis for the resetting of the hypothalamic "gonadostat". This difference between prepuberal and mature gilts in the initial LH response to morphine may reflect differences in the pretreatment serum LH concentrations as well as differences in CNS responsiveness to morphine. Also this difference may be due to an altered rate of metabolism or clearance of morphine as gilts age. Another explanation for this difference may be related to a difference in the size of the ventricular system; i.e., prepuberal gilts may have actually received a larger dose of morphine per ml CSF than mature gilts. Therefore, the ICV cannulated OVX gilt provides an excellent model for further work to delineate whether there is an age related maturation of the opioid-LH axis in the gilt. In conclusion, the results from the present experiment coupled with our previous findings (12) indicate that, in the prepuberal gilt, the EOP receptors are anatomically linked to the GnRH/LH secreting system but that the EOP neurons are not functionally coupled with the GnRH neuronal system.

Acknowledaments The authors thank Ms. Elizabeth Taras and Mr. Bennett Johnson for their technical assistance; Rue1 L. Wilson, Biometrician, Southern Region, ARS, for his statistical advice, and Dr. 0. J. Bolt, USDA, Beltsville, MD, for providing pituitary hormone used in radioimmunoassay. This research was supported by USDA funds and State and Hatch funds allocated to the Georgia Agricultural Experiment Station. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the U. S. Department of Agriculture or the University of Georgia and does not imply its approval to the exclusion of other products which may be suitable.

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