We have shown previously in pubertal boys that testosterone (T) suppresses the nocturnal augmentation of luteinizing hormone (LH) secretion principally by ...
0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society
Vol. 82, No. 12 Printed in U.S.A.
Acute Effects of Estradiol Infusion and Naloxone on Luteinizing Hormone Secretion in Pubertal Boys* G.B. KLETTER, V. PADMANABHAN, I.Z. BEITINS, J.C. MARSHALL, R.P. KELCH, AND C.M. FOSTER Department of Pediatrics (G.B.K., V.P., I.Z.B., R.P.K., C.M.F.) of the University of Michigan, Ann Arbor, Michigan 48105; Department of Medicine (J.C.M.), University of Virginia Health Sciences Center, Charlottsville, Virginia 22908 ABSTRACT We have shown previously in pubertal boys that testosterone (T) suppresses the nocturnal augmentation of luteinizing hormone (LH) secretion principally by decreasing LH pulse frequency. As T can be aromatised to estradiol (E2), and E2 effects on LH secretory dynamics may be separate from those of T, we examined the effects of acute E2 infusion on LH secretion in pubertal boys. Opioid receptor blockade has been reported to increase LH secretion after estradiol suppression in adult men, so we also examined whether naloxone might augment LH secretion during E2 treatment in pubertal boys. Starting at 1000 h, eight pubertal boys were given a 33 h saline infusion, followed 1 week later by an E2 infusion at 4.6 nmol/m2/h. During both infusions, four iv boluses of saline were given hourly beginning at 1200 h on the first day, and four naloxone iv boluses, 0.1 mg/kg each, were given hourly beginning at 1200 h on the second day. Blood was obtained every 15 min for LH, and every 60 min for T and E2, from 1200 h until the end of the infusion. Pituitary responsiveness to gonado-
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UTEINIZING hormone (LH) secretion, and by inference, gonadotropin-releasing hormone (GnRH) secretion have been shown to be responsive to the negative feedback effects of sex steroids at all stages of pubertal maturation (1–10). In adult men, testosterone (T) and dihydrotestosterone (DHT) slow the LH pulse frequency, whereas estradiol (E2) decreases LH pulse amplitude and pituitary responsiveness to exogenous GnRH (6 –10). The nocturnal augmentation of LH secretion that heralds the onset of puberty is relatively resistant to suppression by acute infusion of T, and T in pubertal boys slows the LH pulse frequency with little effect on LH pulse amplitude (4, 5). E2 increases during puberty as T concentrations rise. We tested the hypothesis that E2 infusion in pubertal boys would have similar effects on LH secretion as those seen in adults. Data derived from studies in adult men suggest that the negative feedback effects of sex steroids are mediated via endogenous opioid pathways, as coadministration of an opioid receptor blocking agent reverses these suppressive efReceived March 5, 1997. Revised August 29, 1997. Accepted September 16, 1997. Address all correspondence and requests for reprints to: Gad B. Kletter, M.D., Division of Pediatric Endocrinology, Children’s Hospital of Seattle, P.O. Box 5371, Mail Stop CH-92, 4800 Sand Point Way, Seattle, Washington 98105-5371. * This work was supported by National Institutes of Health Grant HD-16000 and Clinical Research Center Grant M01-RR-00042. The results were presented in part to the 75th Annual Meeting of The Endocrine Society, Las Vegas, Nevada, 1993.
tropin-releasing hormone (GnRH) was assessed after both infusions by iv administration of 250 ng/kg synthetic GnRH. Estradiol infusion increased the mean plasma E2 concentration from 23 6 4 to 46 6 6 pmol/L (P , 0.01) and suppressed mean plasma T from 4.9 6 1.4 to 3.0 6 3.5 nmol/L (saline vs. E2 infusion, P , 0.05). The overall mean LH was suppressed by E2 infusion from 3.7 6 0.5 to 2.2 6 0.4 IU/L (saline vs. E2 infusion, P , 0.01). LH pulse frequency was suppressed by 50%, whereas mean LH pulse amplitude was not different between saline and E2 infusions. Administration of naloxone did not alter the mean LH, LH pulse frequency, or amplitude during either saline or E2 infusions. Pituitary responsiveness to exogenous GnRH was similar during both infusions. These studies indicate that E2 produces its negative feedback in pubertal boys principally by suppression of LH pulse frequency, and naloxone does not reverse these suppressive effects. Thus E2 suppression of LH secretion is mediated by a decrease of hypothalamic GnRH secretion that is independent of endogenous opioid pathways. (J Clin Endocrinol Metab 82: 4010 – 4014, 1997)
fects (8, 9). Regulation of LH secretion by endogenous opioid pathways develops during pubertal maturation, as studies in children and men demonstrate that opioid receptor blockade has little, if any, effect on LH secretion until the later stages of pubertal maturation (8 –17). E2 and T may have differing effects on LH secretion and expression of opioid tone, and thus we determined whether E2 negative feedback effects on LH secretion could be mediated via endogenous opioid pathways by assessing whether the opioid receptor blockade by naloxone could reverse the suppression of LH secretion by E2 infusion. Subjects and Methods Subjects Eight healthy boys were recruited from the Pediatric Endocrine Clinics of the University of Michigan. Before enrollment in the study, each boy had a history and physical examination, pubertal staging by the method of Marshall and Tanner (18), and a bone age determined by the method of Gruelich and Pyle (19). The clinical characteristics of the boys are shown in Table 1. All boys were endocrinologically normal at the time of the study except subject 7, who had exogenous obesity.
Protocol All studies were conducted in the Clinical Research Center of the University of Michigan Hospitals after informed consent was obtained from a parent and assent from the subject. The protocols were approved by the Human Investigation Committee of the University of Michigan. The boys were hospitalized twice, one week apart. Studies were performed on the second night of each admission to allow for acclimatization. Sleep was monitored visually by trained nursing personnel.
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TABLE 1. Clinical characteristics of boys participating in the study Patient no.
Age (yr)
Bone age (yr)a
Ht (cm)
Wt (kg)
Diagnosisb
1 2 3 4 5 6 7 8
13.75 13.83 14.17 14.41 15.17 15.17 15.67 15.83
10 11 11.5 12.5 11 12 14.5 12.5
143.4 142.3 146.0 147.7 141.7 146.3 182.8 149.5
34.1 35.6 49.7 35.2 31.8 32.8 129.0 40.2
CGD CGD CGD CGD CGD CGD Obesity CGD
Pubertal Stagec Pubic hair
II II II II II II III II
Genital
II II III II II II III II
a
Determined by method of Greulich and Pyle (19). Abbreviations used are: CGD, constitutional growth delay. c Determined by the method of Marshall and Tanner (18). b
Pulse analyses Pulse analysis was performed using the Detect program developed by Oerter et al. (21). Peaks were detected using the predicted variance model, with a threshold for false positive peak detection at less than 1%. LH values less than the assay sensitivity were assigned the value of assay sensitivity. Missing values comprised less than 1% of the total samples and were not replaced. LH pulse amplitudes were calculated as the difference between the peak height and the leading nadir value. Pulses were accepted if the amplitude was greater than assay sensitivity.
Statistical analyses
FIG. 1. The experimental design used for the boys is depicted. Lights were turned off at 2200 h. The boys were awakened at 0600 h. On the study day, an indwelling cannula was inserted into a forearm vein of each arm, one cannula for infusion and one for frequent blood sampling. A Harvard infusion pump was used to administer all infusions (Harvard Apparatus, South Natick, MA). A schema for the combined E2 infusion/naloxone boluses study is depicted in Fig. 1. During the first week of the study, patients received an infusion of normal saline starting at 1000 h until 1840 h the following day. E2 infusion (4.6 nmol/m2/h) was given on the second week of the study. The first day of both the saline infusion and the E2 infusion studies was spent acclimatizing to the unit. Starting at 1200 h on the second day of each study, blood samples were drawn every 15 min for LH and every 60 min for T and E2 until 1800 h, and then again from 2200 h until 1800 h on the last day. During both infusions, four intravenous boluses of saline were given hourly beginning at 1200 h on the first day, and four 0.1 mg/kg intravenous boluses of naloxone were given hourly beginning at 1200 h on the second day. At the end of each infusion, pituitary responsiveness to synthetic GnRH (Factrel®, Wyeth-Ayerst, Philadelphia, PA) was assessed by administering an iv bolus of 250 ng/kg at 1800 h followed by blood samples drawn at 1820 and 1840 h.
Hormone measurements Plasma LH was measured in duplicate by RIA as described previously (1, 14, 20) using WHO standard 78 –549. Results are expressed in terms of the Second International Reference Preparation of human Menopausal Gonadotropin. The sensitivity of the assay was 1.0 IU/L, and the intra- and interassay coefficients of variation (CVs) were 4% and 8% respectively. Plasma T and E2 concentrations were determined in duplicate by RIA kits obtained from Radioassay Systems Laboratories, Inc. (Carson, CA). The sensitivity of the T assay was 0.35 nmol/L, and the intra- and interassay CVs were 5% and 8.5% respectively. The sensitivity of the E2 assay was 18 pmol/L, and the intra- and interassay CVs were 8% and 15%, respectively. Where possible, all samples from an individual were analyzed in the same T, E2, or LH assay.
All data were transformed logarithmically to adjust for heterogeneity except for LH pulse frequency data, which underwent square root transformation. Mean plasma hormone concentrations between groups were analyzed by a two-way ANOVA for repeated measures, and, within groups, by a one-way ANOVA for repeated measures. LH pulse frequency was analyzed by Student’s paired t test between treatments, or by Fisher’s weighted least squares analysis within treatments. Differences in plasma LH values over time were determined by regression analysis. The mean incremental LH responses to GnRH after saline and E2 infusions were compared by Student’s paired t test. P , 0.05 was considered significant. Combined data are represented as means 6 se.
Results Effects of E2 infusion on LH pulse frequency and amplitude
Results from subject 8 are shown in Fig. 2. This 15.8 yr boy (bone age 12.3 yr) had an increase in mean plasma E2 from less than 18 pmol/L during saline infusion to 30.5 6 6.0 pmol/L during the E2 infusion. He had an increase in nocturnal mean serum LH (top panel) during the saline infusion (mean LH 5.9 6 1.0), which was suppressed during the E2 infusion (2.0 6 0.5 IU/L). LH pulse frequency was suppressed by the E2 infusion (10 vs. 4 pulses in 26 h). Peak pituitary LH released by exogenous GnRH was 30.2 IU/L during the saline infusion and 23.8 IU/L during the E2 infusion. Data obtained from all eight boys are shown in Fig. 3. The data were analyzed by the following time blocks: 1200 –1800 h on Day 1, the baseline observation period when saline boluses were administered (saline and E2 infusions); 2200 – 0600 h, the night time period (lights turned off); 0615–1145 h, the daytime period (patients awakened at 0600 h); and 1210 –1800 h on Day 2, during which time naloxone boluses were administered. The E2 infusion increased mean serum E2 concentration 2-fold from 23 6 4 to 46 6 6 pmol/L (saline vs. E2 infusion, P , 0.01). The E2 infusion also decreased mean plasma T concentration from 4.9 6 1.4 during saline infusion to 3.0 6 3.5 nmol/L (P , 0.05). The overall mean plasma LH
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Effects of naloxone on LH secretion during saline and E2 infusion
Administration of naloxone did not alter the mean LH, LH pulse frequency, or amplitude during either saline or E2 infusions (Fig. 3). During saline infusion, from 1200 –1800 h, when saline boluses were given, the mean serum LH was 2.6 6 0.5 IU/L, and 3.1 6 0.9 IU/L from 1200 –1800 h the following day when naloxone boluses were given. The following week, when E2 was infused, mean serum LH was 2.1 6 0.4 IU/L from 1200 –1800 h during saline boluses and 1.9 6 0.5 IU/L from 1200 –1800 h the following day, despite the administration of naloxone boluses. Baseline mean LH pulse frequency during saline infusion was 0.33 6 0.08 pulses/boy/h and increased no further (0.29 6 0.08 pulses/ boy/h) during naloxone treatment. With the E2 infusion, baseline LH pulse frequency was 0.23 6 0.09 pulses/boy/h
FIG. 2. Results of an informative 15 yr, 10 mo (bone age 12 yr, 3 mo) boy are shown. The upper panels show the serum LH, T, and E2 concentrations during saline infusion, while the bottom panels show the data during E2 infusion. Asterisks denote significant LH pulses. Arrows indicate administration of saline, naloxone (0.1 mg/kg/bolus), and GnRH (250 ng/kg).
concentration was 3.7 6 0.5 IU/L during the saline infusion and decreased to 2.2 6 0.4 IU/L during E2 infusion (P , 0.01). There was a 50% decrease in the LH pulse frequency with E2 administration, such that, for all the boys, 99 LH pulses were identified during saline infusion as compared with 49 LH pulses during E2 infusion (P , 0.01). Mean LH pulse amplitude was 3.1 6 0.6 IU/L during saline infusion and 2.8 6 0.6 IU/L during E2 infusion (P 5 NS). Nocturnal augmentation of LH secretion was assessed by comparison of results obtained during the baseline time block of 1200 –1800 h to those at 2200 – 0600 h. During saline infusion mean LH was 5.4 6 0.6 IU/L and LH pulse frequency was 0.83 6 0.06 pulses/boy/h between 2200 – 0600 h, compared with 2.6 6 0.5 IU/L and 0.33 6 0.08 pulses/boy/h between 1200 –1800 h (P , 0.01); thus there was a nocturnal increase in LH secretion. During the E2 infusion, mean LH was 2.8 6 0.6 IU/L and the LH pulse frequency was 0.33 6 0.09 pulses/boy/h between 2200 – 0600 h, compared with 2.1 6 0.4 IU/L and 0.23 6 0.09 pulses/boy/h between 1200 – 1800 (P 5 NS), demonstrating that the nocturnal increase of LH secretion was abolished by the administration of E2.
FIG. 3. Mean plasma LH concentrations, LH pulse frequency, and amplitude and mean plasma T and E2 in eight boys who participated in the study. Data are presented as mean 6 SE. Asterisks denote significance (P , 0.05) for saline vs. E2 infusion.
EFFECTS OF NALOXONE AND E2 ON LH SECRETION
and 0.17 6 0.09 pulses/boy/h during naloxone treatment. During saline infusion, baseline LH pulse amplitude was 3.0 6 0.6 IU/L and 2.7 6 0.6 IU/L with naloxone treatment. During the E2 infusion, baseline LH pulse amplitude was 2.2 6 0.5 IU/L, and 3.1 6 0.6 IU/L during naloxone treatment. Effects of E2 on pituitary responsiveness to GnRH
Pituitary responsiveness to GnRH stimulation was determined at completion of the saline and E2 infusions. The mean LH amplitude after 250 ng/kg synthetic GnRH did not differ significantly with treatment (21.2 6 4.1 IU/L during saline infusion vs. 16.9 6 3.0 IU/L during E2 infusion, P 5 0.14). Discussion
This study was designed to determine whether E2 negative feedback control in boys is similar to that seen in adult men. In men, E2 is reported to diminish LH pulse amplitude and pituitary response to exogenous GnRH (9), an effect that may be reversed by opioid blockade (8, 9). In contrast to the studies in adult men, E2 suppresses mean LH concentration in pubertal boys principally by blunting the increase in LH pulse frequency that occurs coincident with sleep. E2 administration does not blunt LH pulse amplitude in these early- to midpubertal boys. Further, E2 does not consistently suppress the amplitude of LH pulses released in response to exogenous GnRH in our pubertal boys. Thus the mechanisms by which E2 suppresses LH secretion are different in boys and men. Our studies indicate that E2 negative feedback occurs primarily by decreasing hypothalamic GnRH secretion in boys, whereas previous studies suggest that E2 blunts pituitary responsiveness to GnRH in adult men (9). Direct comparison of our study to previous studies on the effect of E2 on LH secretion in adult men are complicated by the use of greater doses of E2 or longer duration of administration. It could be that utilization of a larger, less physiologic E2 dose in pubertal boys may produce suppression of pituitary gonadotropin release. Our study has the advantage that physiological concentrations of E2 for adult males were attained. Recently, Wu et al. (22) have demonstrated, using a highly sensitive immunofluorometric assay, that the nocturnal augmentation of LH secretion results from an increase in LH pulse amplitude. They also demonstrated that there is a 2-fold increase in LH pulse frequency, a finding similar to what we observed in this group of boys (Fig 3). In our study, LH pulse amplitude did not show similar changes as reported by Wu et al. (22). The main reason for the differences stem from the way LH pulses have been defined in the two studies. We calculate the amplitude as the peak value minus the nadir just before an identified LH pulse, whereas Wu et al. determine LH plasma concentrations using an assumption that there are no significant interburst tonic secretion events (22). More sensitive LH assays, such as the immunofluorometric assay (Delfia) have been proposed to be able to detect LH pulses with amplitude below the assay detection ability of the RIA. We examined this issue previously and found that the more sensitive LH assay did not result in an increase in the number of LH pulses identified (23). More significant was
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the fact that the physiological interpretation of the effects of T infusion and naloxone on LH pulse frequency and amplitude and the responses to GnRH were not altered whether LH was measured by RIA or immunofluorometric assay (23). The responsiveness of the reproductive system to sex steroids has previously been shown to occur at all ages (2, 4 –7). Thus, it is no surprise that the E2 infusion results in a decrease in LH secretion in these boys. The amount of E2 needed to achieve the suppression was 200-fold less than the amount of T used in our previous studies (10, 17, 26), suggesting that E2 is a much more potent suppresser of LH secretion even in boys. Our previous T infusion studies in boys and men also indicate that a change in the mechanism of sex steroid negative feedback occurs with maturation. Boys exhibit T suppression of hypothalamic GnRH secretion manifested by a decrease in LH pulse frequency but not amplitude, but the hypothalamus becomes resistant to sex steroid negative feedback with puberty (1–5). In contrast, the same dose of T given to adult men decreases pulse amplitude but not pulse frequency, while blunting pituitary sensitivity to GnRH (10). Because DHT administration and alternative T administration regimens have been shown by others to reduce pulse frequency but not amplitude in adult men (9), it is possible that our observed effects of T are related to its aromatization to E2. The important fact to note, however, is that puberty in boys appears to be characterized by increasing resistance of the hypothalamus to sex steroid negative feedback, so that as puberty progresses, sex steroids decrease LH secretion more by decreasing pituitary sensitivity to GnRH and less by suppressing GnRH secretion. We studied the role of E2 and endogenous opioid pathways and their inter-relationship in the regulation of the pulsatile secretion of LH and, by inference, GnRH secretion during pubertal maturation in boys. The results in this group of boys demonstrate that administration of naloxone cannot reverse the suppressive effects of the E2 infusion on LH secretion. Opioid receptor blockade has no effect on LH secretion during either saline or E2 infusion, whereas E2 infusion suppresses mean LH and LH pulse frequency. There is the possibility that not enough naloxone was administered, but this seems unlikely in light of the large dose of naloxone given. Each boy received four iv doses of naloxone; each of these doses was 2.5 times larger than the usual recommended dose for reversal of narcotic overdose (Narcan, Du Pont Pharmaceuticals, Wilmington, DE). Opioid receptor blockade has been shown to increase LH secretion in adult men (8, 9), whereas in prepubertal and early pubertal boys no release of LH can be demonstrated (14 –17, 24, 25). Indeed, a paradoxical suppression of LH by naltrexone has been shown in early pubertal boys (12). Moreover, even children with precocious puberty do not manifest an augmentation of LH secretion following the administration of naloxone (25). Not only have we been unable to disinhibit the suppression of LH secretion by sex steroids, we and others have been unable to show a response of LH secretion to opioid receptor blockade in early puberty, either during the daytime or at night (17, 26). Taken together, these data suggest that the regulation of LH secretion by endog-
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enous opioid pathways develops at the later stages of puberty. The concept that opioid modulation of the reproductive system develops during pubertal maturation is further supported by animal studies. Prepubertal animals from numerous species do not increase LH secretion secondary to opioid receptor blockade, whereas adult animals do (27–36). Similarly, opioid regulation of LH secretion has not been observed in lambs maintained in a prepubertal state by nutritional restriction until the lambs are re-fed and puberty progresses (37, 38). Thus, it appears that the modulation of LH secretion by endogenous opioid pathways develops during the later phases of pubertal maturation. Our studies indicate that E2 suppresses hypothalamic GnRH secretion in pubertal boys by depressing the nocturnal increase in LH secretion through a reduction in LH pulse frequency. Endogenous opioid pathways do not appear to be active in prepubertal and early pubertal boys and are unlikely to mediate sex steroid negative feedback suppression of hypothalamic GnRH secretion. This contrasts with the presumed mechanism of sex steroid negative feedback in adult men, where sex steroids suppress LH pulse amplitude, probably by decreasing pituitary sensitivity to GnRH secretion. The role of E2 in suppression of GnRH secretion and its mediation by endogenous opioids in adult men requires further study. It is apparent from these studies, however, that sex steroid control of LH secretion undergoes maturational change during puberty. Acknowledgments We gratefully acknowledge the assistance of the nursing staff of the Kughn Clinical Research Center, and the Assay Core of the Reproductive Sciences Program of the University of Michigan for the LH antisera and iodinated preparations. We also acknowledge the expert technical assistance of Ms. C.M. Zawacki and Mrs. K.M. Kersey in performing the assays.
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