0013-7227/91/1296-3009$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society

Vol. 129, No. 6 Printed in U.S.A.

A Role for Norepinephrine in the Control of Puberty in the Female Rhesus Monkey, Macaca Mulatto,* ANDREA C. GOREf AND El TERASAWA Wisconsin Regional Primate Research Center and Neuroscience Training Program, University of Wisconsin, Madison, Wisconsin 53715

ABSTRACT. The onset of puberty in female rhesus monkeys is characterized by increases in pulsatile LHRH release. In this study we have tested the hypothesis that changes in input to the LHRH neurosecretory system from noradrenergic neurons contribute to this pubertal increase in LHRH release. In the first experiment, the ability of the LHRH neurosecretory system of prepubertal (12-20 months of age, no signs of puberty evident), early pubertal (24-30 months, premenarchial), and midpubertal (30-45 months, postmenarchial but prior to first ovulation) monkeys to respond to ai-adrenergic stimulation was tested. LHRH release in the stalk-median eminence of conscious monkeys was measured using an in vivo push-pull perfusion method. During push-pull perfusion, perfusates were collected continuously in 10-min fractions, and the ai-adrenergic stimulant methoxamine (MTX, 10~8,10"5 M) or vehicle was infused through the push cannula for 10 min at 90 min intervals. LHRH levels in perfusates were estimated by RIA. Monkeys in all three age groups responded to MTX with significant increases in LHRH

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E HAVE recently reported that pulsatile release of LHRH increases during puberty in the female rhesus monkey (1). This increase is critical to the onset of puberty, since pulsatile infusion of LHRH into sexually immature monkeys resulted in precocious puberty (2). However, the increase in LHRH release at the onset of puberty is probably not due to changes in the properties of the LHRH neurosecretory system itself, since: 1) The number and distribution of LHRH neurons does not undergo much variation from mid-gestation through adulthood (3, 4); 2) LHRH messenger RNA (mRNA) can be detected in the hypothalamus at the first trimester of Received June 3,1991. Address all correspondence and requests for reprints to: Ei Terasawa, Ph.D., Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715. * All experiments presented in this manuscript were performed following the standards established by the Animal Welfare Act and the documents entitled "Principles for Use of Animals and Guide for the Care and Use of Laboratory Animals." The protocol for this study was reviewed and approved by the Research Animal Resource Center, University of Wisconsin. This work (publication 31-005 of the Wisconsin Regional Primate Research Center) was supported by NIH Grants RR00167, HD11355, and T32GM07507. t Present address: Fishberg Research Center in Neurobiology, Mount Sinai Medical Center, New York, New York.

release, with the response of the prepubertal group being significantly greater than that of the older age groups. The results indicate that ai-adrenergic receptors are present and functional prior to puberty. In the second experiment, norepinephrine (NE) release in perfusates collected from monkeys in the three age groups was measured by HPLC with electrochemical detection. NE release increased significantly from the pre- and early pubertal to the midpubertal stage. The enhanced sensitivity of prepubertal monkeys to MTX may be due to the absence of high levels of endogenous NE, which results in a situation similar to denervation hypersensitivity. During the early pubertal stage, increases in input from noradrenergic neurons to the LHRH neurosecretory system may occur, thereby resulting in increases in LHRH release, since early pubertal monkeys are highly sensitive to a-adrenergic input. Therefore, we propose that the increase in NE release during puberty contributes to the developmental increase in LHRH release. {Endocrinology 129: 30093017, 1991)

gestation (5), indicating that the peptide is already synthesized before birth, and expression of LHRH mRNA does not change between the prepubertal period and adulthood in male monkeys (6); 3) LHRH release can be induced prior to puberty by electrical stimulation of the medial basal hypothalamus (7), or chemical stimulation by iV-methyl-D-,L-aspartate (8). These studies indicate that a pool of LHRH exists in the hypothalamus that is not normally released in large amounts until the onset of puberty. Therefore, we hypothesize that changes in input to the LHRH neuronal system from other factors, either hormonal or neuronal, are responsible for the increase in LHRH release during puberty. One neuroactive substance that may contribute to the pubertal increase in LHRH release is norepinephrine (NE). The role of NE in the control of LH and LHRH release in adult animals is well established: NE stimulates LHRH release in adult rhesus monkeys (9) as well as in ovarian-intact or ovariectomized, estrogen-primed rodents (10,11). The overlapping localization of LHRHimmunopositive and tyrosine hydroxylase-positive neurons in rats, cats, and primates (12-15), and reports of direct appositions between tyrosine hydroxylase-positive

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NOREPINEPHRINE AND PUBERTY IN MONKEYS

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and LHRH-positive neurons in rats (15) further suggest a possible role for NE in the control of LHRH release. Moreover, the finding in rats that estradiol accumulates in noradrenergic neurons of the brainstem (16) but not in LHRH neurons (17), indicates that estrogen feedback on LHRH release may occur via noradrenergic neurons. However, to date a role for NE in the control of the onset of puberty is only indirectly indicated from studies demonstrating increases in turnover and content of hypothalamic NE in rats (18, 19), and to our knowledge no studies examining the role of NE in puberty in primates have been reported. The purpose of the first experiment was, therefore, to determine whether the LHRH neurosecretory system of pre- and peripubertal rhesus monkeys is responsive to direct application of an adrenergic agonist. The results would demonstrate whether receptors mediating the effects of NE are located on LHRH neurons and/or interneurons affecting LHRH release at different stages of development. In the second experiment, to determine whether a developmental change in NE release occurs that in turn may contribute to the pubertal increase in pulsatile LHRH release, NE release in the stalk-median eminence of pre- and peripubertal monkeys was measured.

Materials and Methods Animals Female rhesus monkeys (Macaca mulatto) born and raised at the Wisconsin Regional Primate Research Center (Madison, WI) and ranging in age from 12-45 months of age were used in this study. All animals were weaned at 10-11 months of age and housed in pairs with a controlled light cycle of 12 h light: 12 h dark (lights on 0600) and a controlled temperature (22 C). Monkeys were fed a standard diet of Purina Monkey Chow once daily, supplemented with fresh fruit several times per week. Water was available ad libitum. Selection of age groups Monkeys were divided into three stages of pubertal development, based on the following criteria and on those described previously (1, 20, 21): 1) prepubertal (no signs of puberty evident, low levels of circulating LH and estrogen; 17.5 ± 0.4 months of age; n = 11); 2) early pubertal (first sex skin color, increases in circulating LH; premenarchial; 24.6 ± 0.4 months of age; n = 10); 3) midpubertal (postmenarchial but before first ovulation; further increases in LH, estrogen and sex skin color; 37.7 ± 1.0 months of age; n = 12). The date of menarche, and daily observations of menstrual cycles anfl changes in sex skin color were made as described previously (22). Changes in LH, estrogen, and LHRH were also monitored in order to confirm the stage of development for each monkey (1, 22). Implantation of cranial pedestal Monkeys were implanted with a cranial pedestal under halothane anesthesia, as described previously (23, 24). Using x-

Endo • 1991 Vol 129 • No 6

ray ventriculography and a stereotaxic apparatus, the center of the pedestal was placed above the infundibular recess of the third ventricle. The pedestal was attached to the skull with bolts and dental acrylic. Animals were allowed to recover at least 1 month before the initiation of experiments. During this period, monkeys were well-adapted to a primate chair and the experimental environment and investigator before the initiation of experiments, as described previously (24). Implantation of a push-pull cannula Two to three days before a push-pull perfusion experiment, a monkey under ketamine (10 mg/kg BW)/xylazine (2 mg) anesthesia was placed on the stereotaxic apparatus and a cannula (20 gauge outer (pull) cannula and 27 gauge stylet) was inserted in the stalk-median eminence using a hydraulic micromanipulator unit (MO95-B, Narishige, Tokyo, Japan). Placement of the cannula tip in the third ventricle was avoided. The placement of the cannula tip was compared to ventriculograms taken during pedestal implantation as described previously (22, 23). The monkey was placed in a primate chair after the surgery and allowed to recover 2-3 days before the experiment began. Push-pull perfusion Push-pull perfusion experiments were conducted on conscious monkeys. The stylet was replaced with an inner (push) cannula (29 gauge) through which a modified Krebs-Ringer phosphate buffer solution (9) was infused at 22.5 /xl/min by a peristaltic pump (Minipulse3, Gilson Medical Electric, Middleton, WI). Perfusate was collected continuously in 10 min fractions (225 (A) on ice from the outer cannula using an identically calibrated pump. Samples were aliquoted for subsequent measurement of LHRH (150 >Ltl) and catecholamines (approximately 75 n\), and frozen and stored at -70 C. Experimental design Experiment I. Experiments were designed to infuse methoxamine (MTX; Burroughs-Wellcome, Research Triangle Park, NC), an «i-adrenergic agonist, into the stalk-median eminence directly while LHRH release was measured. An aiadrenergic agonist was chosen, since au but not a2 or /?adrenergic agonists and antagonists influenced LHRH release in adult rhesus monkeys (25). Seven prepubertal, six early pubertal, and five midpubertal female monkeys were used. Each animal received two push-pull perfusion experiments: MTX experiment. MTX dissolved in Krebs-Ringer phosphate buffer at a concentration of 10"8 or 10~5 M, or vehicle, was repeatedly infused through the push cannula for 10 min at 90min intervals. MTX at each dose was infused 2 consecutive times except for a few cases in which the protocol was modified due to technical difficulties. Vehicle was infused at the beginning and/or end of the infusion series. Control experiment. Vehicle was repeatedly infused 5-6 times at 90-min intervals. For all experiments, there was a 2- to 3-h control period for stabilization of LHRH release, which was followed by the first MTX or vehicle infusion. The order of the two experiments as well as the order of the two doses was randomized among animals.

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NOREPINEPHRINE AND PUBERTY IN MONKEYS Experiment II. Developmental changes in levels of NE, were determined in perfusates collected during the control period of experiment I as well as in perfusates collected from 15 additional animals at prepubertal (n = 4), early pubertal (n = 4), and midpubertal (n = 7) ages in collateral experiments. For each animal, NE levels in 4-6 samples were measured by HPLC. Aliquots of the same perfusates were analyzed for LHRH levels.

LHRHRIA LHRH in perfusates was measured by RIA (1, 24) using antiserum R1245 which was kindly provided by Dr. T. Nett (26). Synthetic LHRH (Richelieu Laboratory, Inc., Montreal, Canada) was used for the radiolabeled antigen and the reference standard. The antigen-antibody complex was precipitated with a sheep antirabbit-7-globulin. Sensitivity of the assay was 0.1 pg/tube. The intrassay and interassay coefficient of variation were 7.6 and 11.5%, respectively.

Results Effects of MTX or vehicle administration on LHRH release Prepubertal period. MTX (10~8,10~5 M) induced substantial increases in LHRH release in prepubertal monkeys, as shown in two representative cases (Fig. 1, A and B). Prepubertal 87054 18.5 mo

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HPLC NE in aliquots of the same perfusate samples was analyzed by HPLC with electrochemical detection, as described previously (9). Each perfusate sample (~75 ^1) was treated with 10 n\ perchloric acid and centrifuged for 10 min, and 50 n\ of supernatant was injected on a C18 column (ODSII, Regis, Morton Grove, IL) with a mobile phase consisting of 0.17 M acetate buffer, pH 4.8, containing 32 mg/liter sodium octyl sulfate, 3% methanol, and 0.001 M EDTA. The minimum detectable level of NE was 2 pg/sample. Specificity of the NE peak in chromatograms was verified as described previously (9). Samples analyzed by HPLC were collected during the control period of experiments to eliminate the influence of drug infusion on catecholamine levels.

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Statistical analysis LHRH levels in the 20 min before and the four 10-min periods after MTX or vehicle administration were averaged for each age group. Since each dose of MTX was basically administered twice per experiment, means were calculated from two entries per monkey. Control data were calculated from both the MTX experiment in which vehicle had been infused, and from the control experiment in which repeated administrations of vehicle were given. In either experiment data were averaged for each monkey to yield one entry from each experiment. In a few animals, only one entry for a MTX dose or vehicle was made, due to modification of the protocol as described above. Data were analyzed by two-way analysis of variance, followed by Fisher's PLSD test. Significance was attained at P < 0.05. The percent change in LHRH release following MTX or vehicle was calculated for each age group. The mean LHRH value prior to MTX or vehicle infusion (-20-0 min) for each group was designated as 100%, and the peak response relative to this value (from the first two fractions after MTX or vehicle) was calculated accordingly. Developmental changes in NE and LHRH release were compared using one-way analysis of variance followed by Fisher's PLSD test. Each animal was represented by the average of NE or LHRH values. Due to large variances within groups, a BOXCOX transformation was made before analysis (27). Again, significance was attained at P < 0.05.

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FIG. 1. Effects of MTX or vehicle infusion on LHRH release during push-pull perfusion in three representative monkeys at the prepubertal stage. A and B, MTX at concentrations of 10~8 and 10"6 M, as well as vehicle were infused; C, vehicle was infused repeatedly. Vertical bars in this and subsequent figures indicate the 10 min fractions which were exposed to MTX or vehicle.

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NOREPINEPHRINE AND PUBERTY IN MONKEYS

Although in these cases (Fig. 1, A and B) the effect of MTX on LHRH release appeared to diminish with repeated applications, for the group there was no significant effect of repeated exposure to MTX on the magnitude of the LHRH response. Likewise, there was no significant effect of the order of infusion of the two doses of MTX on LHRH release. Repeated vehicle administration in control experiments also did not affect LHRH release (Fig. 1C). Analysis of variance indicated a significant effect of treatment on LHRH release in prepubertal monkeys (P < 0.001). Post hoc analysis indicated that 10~8 and 10"5 M MTX induced highly significant increases in LHRH release (P < 0.001; Fig. 2), while vehicle caused no effect. Administration of MTX (10~8 and 10"5 M) resulted in increases in LHRH release of 629 and 357%, respectively, from the pre-MTX value to the peak at 10 to 20 min, while the change in LHRH induced by vehicle infusion was 94%. Early pubertal period. MTX (10~8, 10~5 M) administration resulted in large increases in LHRH release in early pubertal monkeys, as seen in two representative cases (Fig. 3, A and B). There was no consistent effect of repeated MTX infusion or the order of infusion of the two doses of MTX on LHRH release. Again, vehicle administration did not affect LHRH release (Fig. 3C). Analysis of variance indicated that there was a significant effect of treatment on LHRH release in this age group (P < 0.001). Post hoc analysis indicated that the increases in LHRH release following each dose of MTX (10~8,10"5 M) administration were significant (P < 0.01, 0.05, respectively; Fig. 4). MTX (10~8, 10~5 M) induced

Endo • 1991 Voll29«No6

increases of 332 and 410%, respectively, from before MTX until the peak response after MTX, while vehicle infusion resulted in a change in LHRH release of 130%. Midpubertalperiod. MTX (10~8,10~5 M) infusion induced significant increases in LHRH release, as shown in two representative cases (Fig. 5, A and B), while repeated vehicle administration did not affect LHRH release (Fig. 5C). Again, no consistent effects of repeated MTX infusion or the order of infusion of the two MTX doses on LHRH release were detected. Analysis of variance indicated a significant treatment effect for midpubertal monkeys (P < 0.01). Although the increases in LHRH release in response to both doses of MTX were small, they were significant (P < 0.01; Fig. 6). LHRH release increased 244 and 176% following MTX (10~8, 10"5 M, respectively), while administration of vehicle resulted in a change of 98%. Age effects There was a significant age effect in the response to MTX (P < 0.01). The increase in LHRH release induced by infusion of MTX (10~8 M) in the prepubertal group was significantly greater than that induced in early and midpubertal monkeys (P < 0.01). MTX (10~5 M) induced significantly greater increases in the early pubertal group than either the prepubertal or midpubertal groups (P < 0.05 and 0.01, respectively). Norepinephrine release Overall there was a significant developmental increase in NE release (P < 0.05). The post hoc analysis indicated

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FIG. 2. LHRH release in prepubertal monkeys (n = 7) in the 20 min period before and 10 min periods after MTX or vehicle. MTX (10~8, 10~6 M) infusion resulted in significant increases in LHRH release, while vehicle infusion had no effect on LHRH release. *, P < 0.05 us. -20 to 0; ***, P < 0.001 vs. -20 to 0.

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NOREPINEPHRINE AND PUBERTY IN MONKEYS Early Pubertal A

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Table 1). The developmental increase in LHRH release was similar to that of previous reports (1, 7).

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FiG. 3. Effects of MTX and vehicle infusion on LHRH release during push-pull perfusion in three representative monkeys at the early pubertal stage. A and B, MTX at concentrations of 10~8 and 10"5 M as well as vehicle were infused; C, vehicle was infused repeatedly.

that mean NE release in perfusates obtained from animals at the midpubertal period was higher than that at the prepubertal (P < 0.05) and early pubertal period (P < 0.05, Table 1). There was also a developmental increase in LHRH release (P < 0.01). Mean LHRH release in aliquots of the same perfusate samples increased significantly from the prepubertal to early pubertal (P < 0.05) and early pubertal to the midpubertal stage (P < 0.05,

The present study has demonstrated that the LHRH neurosecretory system of prepubertal monkeys is highly sensitive to the administration of the ai-adrenergic agonist MTX. Infusion of MTX to the stalk-median eminence resulted in significant increases in LHRH release in prepubertal monkeys. This is the first demonstration that «i-adrenoceptors on LHRH neurons or on interneurons contacting LHRH neurons are already present and functional long before the onset of puberty. Early pubertal and midpubertal monkeys also responded to infusion of MTX with significant increases in LHRH release, although the magnitude of the responses decreased with pubertal development. This change in responsiveness to MTX during puberty may be due to a number of possibilities, including effects of circulating steroids, changes in neural circuitry, differences in the properties of the receptors, or other developmental changes, which will be discussed below. The results of the present study indicate that, like LHRH release, NE release is low in prepubertal monkeys, and increases during puberty. It has been reported in rats that a significant increase in synaptogenesis, and specifically catecholamine immunofluorescence in the region around the third ventricle occurs during pubertal development (28-30). Similar anatomical studies have not yet been reported in rhesus monkeys; however, it is possible in this species that developmental increases in catecholaminergic input and synaptogenesis in the hypothalamus-median eminence also occur, resulting in the pubertal increase in NE release as observed in the present study. In addition to the finding that levels of NE released at neuroterminals are low prior to puberty, it is possible that the pattern of NE release is immature prior to puberty. In adult monkeys, NE release in the stalkmedian eminence is pulsatile, and pulses of NE occur synchronously with those of LHRH (9). Prior to puberty, NE release may not yet be pulsatile, or may be asynchronous with LHRH pulses. Furthermore, in adults, NE appears to play a role in amplifying pulses of LHRH, since administration of the ai-adrenergic antagonist prazosin significantly reduced the amplitude of LHRH pulses, without affecting their frequency (25). At the onset of puberty, therefore, an increase in NE release may occur, resulting in an amplification of LHRH pulses. Although the present study did not determine the pattern of NE release, the development of a pulsatile pattern of NE release, or the synchronization of NE with LHRH pulses, may be important for puberty.

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FIG. 4. LHRH release in early pubertal monkeys (n = 6) in the 20-min period before and 10-min periods after MTX or vehicle. MTX (10~8,10~6 M) infusion resulted in significant increases in LHRH release while the infusion of vehicle did not affect LHRH release significantly. *, P < 0.05 vs. -20 to 0; •*, P < 0.01 vs. -20 to 0.

The finding that prepubertal monkeys responded to infusion of MTX with large increases in LHRH release may be due to the fact that their a r adrenergic receptors may be highly sensitive to MTX under low levels of endogenous NE release. This is similar to the situation of denervation hypersensitivity (31), in which receptors that are exposed to little or no endogenous ligand are up-regulated. Under these conditions, application of MTX would be expected to induce large increases in LHRH release in prepubertal monkeys because of the presence of large numbers of unoccupied receptors. Nevertheless, in prepubertal monkeys, the low levels of endogenous NE release in the hypothalamus of prepubertal monkeys may not be sufficient to stimulate LHRH release. The results of the present study also indicate that the responsiveness of the LHRH neurosecretory system to MTX decreases during puberty. While MTX induced significant increases in LHRH release in midpubertal monkeys, these responses were significantly lower than those of prepubertal and early pubertal monkeys. This finding, although unexpected, can be explained in light of the fact that NE release increases during puberty. Since in midpubertal monkeys the higher levels of endogenous NE release may desensitize ai-adrenergic receptors in the hypothalamus, stimulation of the «i-adrenergic receptors with MTX would be expected to have little effect on LHRH release. In the present study, prepubertal monkeys responded to infusion of MTX with robust increases in LHRH release, although their circulating estradiol levels are minimal (22). Similarly, adult ovariectomized monkeys

which are without the influence of estradiol also responded to MTX with significant increases in LHRH release (9). These studies suggest that in primates, the presence of steroids is not obligatory for a stimulatory effect of NE on LHRH release. The lack of steroiddependence is in contrast to studies on the rat and rabbit (10,11,32,33), in which a-adrenergic agonists stimulated LH and LHRH release in ovarian-intact or ovariectomized, estrogen-primed animals, but did not stimulate LHRH release in unprimed, ovariectomized animals (32, 33). The reasons for this species difference are unclear, but it is speculated that neuronal circuitry, localization of estrogen receptors, or route of administration of adrenergic agonists and antagonists may be different. The timing of the onset of puberty in nonhuman primates does not seem to be limited by maturation of LHRH neurons themselves: the distribution and number of LHRH neurons in prepubertal monkeys are similar to those observed in adult monkeys (34); hypothalamic content of LHRH did not change through postnatal development (35); the expression of cellular LHRH mRNA detected by in situ hybridization did not undergo any developmental changes (6). Moreover, the releasable pool of LHRH in neuroterminals of prepubertal, early pubertal, and midpubertal monkeys appears to be quite comparable, since the amount of LHRH release induced by electrical stimulation (7) and by JV-methyl-D,L-aspartate (8) did not differ before and after the onset of puberty. The low levels of LHRH release in prepubertal monkeys (1, 7) (Table 1) must then be due to an immaturity of other neuronal inputs controlling pulsatile LHRH release. The present study suggests that changes

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NOREPINEPHRINE AND PUBERTY IN MONKEYS Midpubertal 86049

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FIG. 5. Effects of MTX or vehicle infusion on LHRH release during push-pull perfusion in three representative monkeys at the midpubertal stage. A and B, MTX at concentrations of 10~8 and 10~6 M as well as vehicle were infused; C, vehicle was infused repeatedly.

in a-adrenergic input to the hypothalamus may be a part of the regulatory mechanism for puberty in female rhesus monkeys. Although NE appears to play a role in the control of the onset of puberty, it is unknown whether this role of NE is mandatory or merely permissive. It has been reported that transections of noradrenergic pathways arising from the brainstem and projecting to the hypo-

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thalamus, or lesions of the locus coeruleus, the source of much of the hypothalamic NE (36) disrupted estrous cyclicity in adult rats only temporarily (37,38). Similarly, in female rhesus monkeys, deafferentation of the medial basal hypothalamus, which disrupts all noradrenergic inputs, did not prevent the normal onset of puberty (39). Moreover, administration of prazosin, an ai-adrenergic antagonist, reduced LHRH pulse amplitude but not pulse frequency (25). In the case of puberty, depletion of NE with the selective neurotoxin DSP4 prior to puberty significantly delayed but did not prevent the onset of puberty in rats (40). It appears, then, that NE plays an important role in the control of puberty and LHRH release, but that other neuronal systems may compensate for the actions of NE when the NE neuronal system is functionally removed. The same is likely true in adults, in which numerous neurotransmitters and neuropeptides play a role in the control of LHRH release (41). Thus, it is quite possible that neurotransmitters and neuropeptides that modulate pulsatile LHRH release in adults, such as NE, neuropeptide Y (42), glutamate (8), or endogenous opiate peptides (43), that modulate pulsatile LHRH release in adults, are all involved in the mechanism of puberty, but they may not be able to function independently to trigger puberty. This concept is supported by the finding that opiate agonists and glutamate antagonists, which are inhibitory to LHRH release, delayed but did not prevent the onset of puberty in female rats (44, 45). Alternatively, the interactions of these substances with one another, or the summation of the actions of these substances, may be critical for the control of puberty. For example, like NE and LHRH, the release of neuropeptide Y at neuroterminals in the stalkmedian eminence increases during puberty (46). The simultaneous increases in NE and neuropeptide Y release, and changes in responsiveness to noradrenergic and neuropeptide Y input, may together result in a stimulation of LHRH release during puberty. In summary, the present study demonstrates that the LHRH neuronal systems of pre- and peripubertal rhesus monkeys respond to administration of MTX, an a^adrenergic agonist, with significant increases in LHRH release. Thus, prior to puberty, ai-adrenoceptors are already present on LHRH neurons or on interneurons affecting LHRH release. In prepubertal monkeys, NE release at the stalk-median eminence is low, and is probably not sufficient to stimulate LHRH release. During the early pubertal period a transition may occur in which NE release begins to increase. Since the LHRH neurosecretory system of early pubertal monkeys is quite sensitive to administration of MTX, this probably results in a developmental increase in LHRH release, which in turn results in the occurrence of menarche. Then, during the midpubertal period, further increases in NE release,

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20 to 30

30 to 40

10 to 20

20 to 30

30 to 40

Time (min)

FIG. 6. LHRH release in midpubertal monkeys (n = 5) in the 20-min period before and 10-min periods after MTX or vehicle. MTX (10~8, 10" M) induced small but significant increases in LHRH release, while vehicle infusion did not affect LHRH release. **, P < 0.01 us. -20 to 0. TABLE 1. Mean NE and LHRH levels in aliquots of the same samples in monkeys at the three stages of puberty. Results are expressed as the mean ± SEM for each age group. Age group

Age (months)

Mean NE (ng/ml/10 min)

Mean LHRH (pg/ml/10 min)

Prepubertal (n = ll) Early pubertal (n = 10) Midpubertal (n = 12)

17.7 ± 0.6

0.33 ± 0.07

0.86 ± 0.24

26.4 ± 0.8

0.36 ± 0.06

2.05 ± 0.44°

35.4 ± 1.1

2.13 ± O.970'6

4.16 ± 1.04*'C

° P < 0.05 vs. prepubertal. b P < 0.05 vs. early pubertal. c P < 0.01 vs. prepubertal.

and subsequently greater increases in LHRH release, occur, ultimately resulting in first ovulation. We believe that developmental changes in the release of and sensitivity to NE, as well as to other neurotransmitters and neuropeptides, contribute to the pubertal increase in LHRH release.

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A role for norepinephrine in the control of puberty in the female rhesus monkey, Macaca mulatta.

The onset of puberty in female rhesus monkeys is characterized by increases in pulsatile LHRH release. In this study we have tested the hypothesis tha...
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