Although. the role of melatonin in humans has not. vet been clarified, there are observations suggest

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1 Effect of an acute injection of melatonin on the basal secretion of hypophyseal hormones in prepubertal and pubertal healthy subjects P. Lisoni,. Resentini, R. auri, C. De edici,. orabito, D. Esposti, L. Di Bella, G. Esposti, D. Rossi, L. Parravicini, G. Legname and. raschini Italian Auxological Centre ofpiancavallo, ilan, Italy Institute ofhuman Physiology1, aculty ofedicine, University ofilan, Italy Chair ofphysiology2, University ofodena, Italy and Chair ofchemotherapy3, aculty ofedicine, University ofilan, Italy Abstract. It is well known that the pineal gland can modulate the secretion of pituitary hormones. elatonin, the main hormone produced by the pineal gland, acts at the hypothalamic site, whereas hypophyseal sensitivity to melatonin seems to change with age. To investigate the influence of pubertal development on the role of the pineal gland in the regulation of the secretion of pituitary hormones, SH,, Prl, TSH and responses to melatonin were evaluated in a group of 9 of both prepubertal and pubertal healthy subjects sexes. elatonin was given im at a dose of 0.2 mg/kg body weight at 3 p.m. Venous blood samples were drawn \m=-\20,0, 20, 40, 60, 90, 120, 180 and 240 min, after melatonin injection. According to the same experimental protocol, venous blood samples were collected during a saline infusion on a separate occasion. SH,, Prl, TSH and plasma levels were measured with RIA. In pubertal subjects, a significant rise in the mean Prl levels was seen 90 min after melatonin as compared with those during saline infusion. The Prl melatonin response area was significantly lower in prepubertal treated subjects and significantly higher in pubertal ones compared with the respective controls. The mean values showed a significant decrease 120 min after melatonin only in prepubertal subjects; no significant variations were seen in 8 of pubertal subjects, whereas in the last 2 a marked increase was observed. inally, under these conditions, melatonin did not influence the basal SH and TSH levels. These results seem to suggest that hypophyseal hormone reponses change with pubertal development. Although the role of melatonin in humans has not vet been clarified, there are observations suggest ing that it is involved in the regulation of the release of pituitary hormones and other endocrine functions. The action of melatonin administration on the hypothalamic-hypophyseal axis has been investigated in only a limited number of human studies. Prolonged administration of melatonin de creases basal blood levels, whereas SH release does not seem to be affected (Nordlund & erner 1977). Nevertheless, other authors did not observe the inhibitory effect of melatonin on gonadotrophin secretion in man (ideleff et al. 1976; Weinberg et al. 1980). Acute melatonin administration suppresses response to insulin hypoglycaemia (Smythe & Lazarus 1974; Smythe et al. 1976). oreover, mela tonin has been shown to suppress L-tryptophanstimulated secretion, wheras it does not inhibit apomorphine-stimulated release (Koulu & Lammintausta 1979). function and cortisol secretion have Thyroid been found to remain unchanged in man during chronic melatonin administration (Nordlund & Lerner 1977). On the other hand, other studies (Jones et al. 1976) revealed an inhibitory action of melatonin on CR release. Downloaded from Bioscientifica.com at 11/19/ :09:47P

2 As far as the effect of melatonin on Prl secretion is concerned, the results are contradictory in man, whereas a stimulatory action has been reported in rats (Kamberi et al. 1971). To further study the action of exogenous mela tonin on the release of pituitary hormones and to investigate whether the hypophyseal response to melatonin changes with pubertal development, the effects of melatonin administration on the basal hormones were studied in a levels of pituitary group of prepubertal and pubertal healthy subjects of both sexes. aterials and ethods Nineteen healthy subjects were studied, 9 of whom were prepubertal (4 males and 5 females), aged between 9.2 and 11.6 years (mean age 9.9 years) and were pubertal (4 males and 6 females), aged between 15.8 and 21.6 years (mean age 17.3 years). The pubertal stage was determined by pubic hair and breast development in the girls according to the Tanner criteria (Tanner 1978). In the boys, pubic hair and Table I. Clinical data for subject groups studied. Subject No. Sex Age (years) Weight (kg) Prepubertal subjects (Tanner stage I) I I- I Pubertal-adult subjects (Tanner stage V) II I I- I Table 2. Areas under the curves, after melatonin administration (0.2 mg/kg body weight, im) and during saline infusion, in prepubertal and pubertal subjects. SH elatonin Prl elatonin elatonin TSH elatonin elatonin Prepubertal X ± SD ± Pubertal X ± SD ± ± ± ± ± ± ± * ± ** ± ± ± ± ± ± ±7.08** ± ± ± *P < 0.05 vs controls. **P < vs controls. genital Tanner staging was performed, and in addition the testicular volume was measured. All clinical measure ments were made by one observer. All prepubertal subjects were at Tanner stage I, whereas the pubertal ones were at Tanner stage V. Women were studied during the menstrual or premenstrual period of their menstrual cycle. The study protocol was explained to the subjects or their parents, and informed consent was obtained. The clinical data are reported in Table 1. elatonin was given im at a dose of 0.2 mg/kg body weight in the afternoon (at 3 p. m.), which is the period of the day when the biological systems are most sensitive to melatonin (Tamarkin et al. 1976). Venous blood were samples obtained from an indwel ling catheter placed in an antecubital vein 20, 0, 40, 60, 90, 120, 180 and 240 min after melatonin injection. According to the same experimental protocol venous blood samples had been collected on the preceding day during a saline infusion only. The whole study was performed during the month of ebruary. Plasma and sera were separated by centrifugation and stored at 20 C until assayed. In each subject, plasma levels of SH,, Prl, TSH and were measured by a double-antibody RIA method using commercially avail able kits (Biodata, Rome). Serum melatonin levels were determined by the RIA method described by Wetterberg et al. (1978) using commercially available kits (WHB, Sweden). The data were analyzed by Student's i-test and the areas under the curves (AUC) were calculated and ana lyzed by a paired Student's i-test. Downloaded from Bioscientifica.com at 11/19/ :09:47P

3 - SH m I U/ml ELATONIN SH ml U/ml ELATONIN o m ins ig. la. SH plasma levels (mean values ± so) after melatonin _J_I-I_L_ ig. lb. SH plasma levels (mean values ± sd) after melatonin injection in pubertal subjects. Results All subjects reported relaxation and sleepiness after the melatonin injection. No undesirable ef fects were observed. SH, Prl, TSH and levels (mean ± SD) after melatonin injection in prepubertal subjects are illustrated in igs, la, 2a, 3a, 4a and 5a, those found in pubertal ones in igs, lb, 2b, 3b, 4b and 5b. The melatonin response areas are listed in Table 2. miu/ml ELATONIN - -RESPONDER CASE melatonin \ -RESPONDER CASE \ saline V miu/ml 3 ELATONIN o 20 1 \.-'" V' V, ig. 2a. plasma levels (mean values ± sd) after melatonin _1_ -1-1_1_1- ig. 2 b. plasma levels (mean values ± sd) after melatonin injection in 9 non-responding pubertal subjects and in 1 responding girl (case No. 19). Downloaded from Bioscientifica.com at 11/19/ :09:47P

4 PRL ELATONIN PRL ELATONIN ig. 3 a. Prl plasma levels (mean values ± SD) after melatonin * < versus controls mins ig. 3 b. Prl plasma levels (mean values ± SD) after melatonin injection in pubertal subjects. SH The mean SH plasma values after melatonin treatment showed no significant differences in comparison to those observed during saline infu sion in the prepubertal subjects nor in the pubertal ones (igs. la,b). No significant variation in the plasma values were seen after melatonin injection in the pre pubertal subjects nor in 9 of the pubertal subjects. On the other hand, in the last subject (a pubertal female, 19.3 years old, in menstrual period, case No. 19), a marked increase of plasma levels was observed 20 min after the mela tonin injection (igs. 2a,b). Prl The melatonin response area was significantly lower (P < 0.05) than that during saline infusion in prepubertal subjects. In contrast, compared with the values obtained during saline infusion, a sig nificant rise in mean Prl plasma values was found in all pubertal subjects 90 min after melatonin injec tion (P < 0.001) (igs. 3a,b). TSH U/ml ELATONIN o TSH µ U/ml 2 ELATONIN o 1,5 0,5 ig. 4a. TSH plasma levels (mean values ± so) after melatonin ig. 4b. TSH plasma levels (mean values ± so) after melatonin injection in pubertal subjects. Downloaded from Bioscientifica.com at 11/19/ :09:47P

5 - 4 3,5 3 2,5 ELATONIN ELATONIN CASE 1 after melatonin CASE 2 after melatonin 2 1, , *p<0 001 versus controls mins ig. 5a. plasma levels (mean values ± sd) after melatonin 2 - The melatonin response are was significantly higher (P < 0.001) in pubertal treated subjects than in the controls (Table 2). TSH The mean TSH plasma levels and the response area after melatonin treatment did not differ sig nificantly from those observed during the saline infusion in the pubertal subjects nor in the prepu bertal ones (igs. 4a,b). A significant decrease of the mean plasma levels (P < 0.001) was seen in prepubertal subjects 120 min after melatonin injection as compared with that observed during saline infusion. In con trast, melatonin did not affect the mean plasma levels in 8 of pubertal subjects, whereas in the last 2 (both males, cases No. 12 and 17), was strongly stimulated by melatonin, with a peak at 40 and 120 min, respectively (igs. 5a,b). In prepubertal treated subjects, the melatonin response area was significantly lower (P < 0.001) than during saline infusion (Table 2). ig. 5b. plasma levels (mean values ± SD) after melatonin injection in 8 non-responding pubertal subjects and in 2 responding boys (cases Nos. 12 «and 17 O-o). elatonin After melatonin injection, the melatonin serum concentrations were at least 0 times greater than the saline infusion. during Table 3 shows the mean melatonin serum levels during saline infusion and after melatonin injec tion. Discussion The results of this investigation suggest that acute administration of melatonin can affect the basal levels of and Prl only: was inhibited by melatonin in the prepubertal subjects only, where as Prl was stimulated in the pubertal subjects and inhibited in the prepubertal ones. Downloaded from Bioscientifica.com at 11/19/ :09:47P

6 elatonin serum levels (pg/ml ± 4.2 Table 3. mean ± sd) after an im injection of 0.2 mg/kg body weight of melatonin and during saline infusion in 9 prepubertal and in pubertal-adult healthy subjects. elatonin (pg/ml) times (min) ISO ± 6.2 ± ± ± ± ± ± ±9.4 elatonin ±5.6 ±6.2 ±56.9 ±114.2 ±396.4 ±417.2 ±321.9 ±226.9 ±191.3 Our data do not confirm the melatonin stimula tory effect on secretion described by Smythe & Lazarus (1975) in adult healthy subjects, since rose after melatonin administration in only 2 of the 19 subjects investigated. On the other hand, a significant decrease of the levels was seen in the prepubertal subjects. elatonin seems to act on the central control of release by blocking serotoninergic transmis sion (Koulu & Lammintausta 1979), and moreover, it can block secretion stimulated by i.-dopa (Nordlund & Lerner 1977). Since melatonin may cause either an elevation of brain serotonin con centration (Anton-Tay et al. 1968) or a blockade of serotoninergic receptors, its ability to enhance basal levels mav be due to increased brain serotonin content, whereas their inhibition may be caused by a blockade of serotonin receptors. The different results described by various au thors on Prl response to melatonin may be ex plained by several melatonin-induced neuroendo crine effects. Data presented in this study point at a melatoninstimulating action on basal Prl levels in pubertal subjects and an inhibitory effect in the prepubertal ones, which is in contrast to the observations by Smythe et al. (1976) who reported absent response of blood Prl levels in man after oral melatonin administration. In agreement with the data previously described in man by ideleff et al. (1976) and by Weinberg et al. (1980), our results do not allow for concluding on any inhibitory effect of melatonin on SH and secretion in prepubertal and pubertal subjects. On the contrary, the increase after melatonin injection observed in one girl during menstrual bleeding could show that melatonin in some condi tions may play also a stimulatory role on gonado trophin secretion. The inhibitory effect of pro longed treatment with melatonin on release referred by Nordlund & Lerner (1977) suggests that different doses and chronic administration of to affect the basal melatonin are probably required secretion of SH and. It has been observed in animals (Reiter 1981) that melatonin can either induce or prevent gonadal atrophy. The contradictory results reported by various authors about melatonin effects of go secretion mav be due to different nadotrophin investigation conditions. In fact, several factors such as photoperiod length, doses, time and way of administration during the day influence the effect of melatonin; moreover, its biological activity de pends not only on its plasma levels, but also on the sensitivity of the target organ; the latter factor represents a great drawback for clinical studies with melatonin. TSH secretion does not seem to be influenced by acute administration of melatonin at the dose chosen in this study. Our results show that there is a difference in and Prl responses to melatonin between prepuber tal and pubertal subjects, and they seem to suggest that the effects of melatonin on hypophyseal hor mone secretion change with age also in man, and that sexual development influences the sensitivity of the hvpothalamic-pituitary axis to melatonin action. It is known that melatonin affects hypo physeal hormone release by acting centrally at the hypothalamic site, whereas the sensitivity of the hypophysis to melatonin, at least in rats (Wurtman et al. 1964), is an age-related process, since in Downloaded from Bioscientifica.com at 11/19/ :09:47P

7 neonatal (but not in adult) pituitaries melatonin inhibits the stimulation of SH and induced by LRH. Human pituitary sensitivity to melatonin has not yet been well investigated, and it is unknown whether it is an age-related process. Showing different responses of and Prl to melatonin in prepubertal and pubertal subjects, the results of this study could suggest that the sensitivity of the hypophysis to melatonin changes with age also in humans; sexual maturation could decrease the pituitary sensitivity to melatonin, and this mav explain the different responses observed between prepubertal and pubertal subjects. We can only hypothesize that the net effects of melatonin on pituitary hormone basal levels re present a combination of at least two actions, the former at the hypothalamic site and the latter at the hvpophyseal one. Therefore, the changes in the sensitivity of the hypophysis with puberty could explain the differ ent responses of and Prl between prepubertal and pubertal subjects. The data of the present study do not allow us to say whether the observed effects of melatonin on pituitary hormones are pharmacological or whether they reflect a physiological role. ore over, the effects of exogenous melatonin may dif fer from those induced by the endogenous hor mone, since the pineal gland itself is a target organ for exogenous melatonin (Wurtman et al. 1964), which could modulate the secretion of the endo genous hormone or down-regulate its receptors. urther studies, during different photoperiods of the day, using either higher or lower doses of melatonin and a prolonged treatment and evaluat ing the basal and stimulated pituitary hormone secretions, are needed to clarify the role of mela tonin in the regulation of hvpophyseal hormone release in humans. References Anton-Tay, Chou C, Anton S & Wurtman R J (1968): Brain serotonin concentration: elevation following intraperitoneal administration of melatonin. Science 162: ideleff H, Aparicio J, Guitelman A, Debeljuk L, ancini A & Cramer C (1976): Effect of melatonin on the basal and stimulated gonadotropin levels in normal men and women. postmenopausal J Clin Endocrinol etab 42: Jones T, Hillhouse E 8c Burden J (1976): Secretion of corticotropin releasing hormone in vitro. In: artini L 8c Ganong W (eds). rontiers in Neuroendocrino logy, vol 4: p Raven Press, New York. Kamberi I A, ical R S & Porter J C (1971): Effects of melatonin and serotonin on the release of SH and prolactin. Endocrinology 88: Koulu & Lammintausta R (1979): Effect of melatonin on I.-tryptophan- and apomorphine-stimulated growth hormone secretion in man. J Clin Endocrinol etab 49: Nordlund J 8c Lerner A (1977): The effects of oral melatonin on skin color and on the release of pituitary hormones. J Clin Endocrinol etab 45: Reiter R J (1981): Reproductive effects of the pineal gland and pineal índoles in the Syrian hamster and the albino rat. In: Reiter R J (ed). The Pineal Gland. Reproductive Effects, vol 2, 45. CRC Press, Boca Raton. Smythe G A & Lazarus L (1974): Suppression of human growth hormone secretion by melatonin and cyproheptadine.j Clin Invest 54: Smythe G A & Lazarus L (1975): Growth hormone responses to melatonin in man. Science 184: Smythe G A, Compton J & Lazarus L (1976): The actions of I.-dopa and 2-bromo-a-ergocryptine. In: Pecile A 8c üller E (eds). Growth Hormone and Related Peptides, 222. American Elsevier, New York. Tamarkin L, Westrom W K, Hamill A I & Goldman D (1976): Effects of melatonin on the reproductive sy stems of male and female Syrian hamsters. A diurnal rhythm in sensitivity to melatonin. Endocrinology 99: Tanner J (1978): oetus into an. Harvard University Press, Cambridge. Weinberg U, Weitzman E D, ukushima D K, Cancel G 8c Rosenfeld R (1980): elatonin does not suppress the pituitary luteinzing hormone response to luteiniz ing hormone-releasing hormone in men. J Clin Endo crinol etab 51: Wetterberg L, Eriksson O, riberg Y & Vangbo (1978) : A simplified radioimmunoassay for melatonin and its application to biological fluids. Preliminary observa tions on the half-life of plasma melatonin in man. Clin ChimActa86: Wurtman R J, Axelrod J & Potter L (1964): The uptake of [3H]melatonin in endocrine and nervous tissues and the effects of constant light exposure. J Pharmacol ExpTher 143: Received on arch 4th, Downloaded from Bioscientifica.com at 11/19/ :09:47P