E E. concentration fell lower than 1 ng/ml. The amplitude was equal to the maximal value of the rise minus the mean concentration

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1 BOLOGY OF RPRODUCTON 52, 56-6 (995) Regulation of Luteinizing Hormone-Releasing Hormone (LHRH) Secretion by Melatonin in the we.. Changes in N-Methyl-D,L-aspartic acid-induced LHRH Release during the Stimulation of Luteinizing Hormone Secretion by Melatonin' CATHRN VGU, 3 ALAN CARATY, ALAN LOCATLL, and BNOT MALPAUX 2 Laboratoire de Neuroendocrinologie Sexuelle, NRA-PRMD, Nouzilly, France ABSTRACT njection of N-methyl-D,L-aspartic acid (NMDA), a glutamatergic agonist, causes an acute release of LH. The amount of LH released is modulated by photoperiod in sheep; it is larger in animals in which reproductive activity has been inhibited by long days than in those photostimulated by short days. The purpose of the present experiment was to determine whether this effect of photoperiod on LH response to NMDA is mediated by melatonin and whether it reflects a change in the amount of LHRH released in response to NMDA. Twenty-one ovariectomized ewes, bearing an s.c. implant of estradiol and kept in long days (6L:8D), received s.c. implants of melatonin on Day 0. The effect of a single injection of NMDA (5 mg/kg i.v.) on LHRH and simultaneous LH secretion was studied at three different periods of the stimulation of LH secretion by the melatonin treatment: first, during inhibition of LH secretion by long days (on Day -, n = 6); secondly, during the onset of the LH increase (on Day 39, n = 9); and thirdly, during maximum LH secretion (on Day 74, n = 6). The stimulation of LHRH secretion by administration of NMDA was characterized by a large and long increase in LHRH concentration on Day - and Day 39. There was no significant difference between these two periods in terms of duration and amplitude of the LHRH release. On Day 74, the stimulation was much shorter than on Day - and Day 39 and was followed by a phase of decreased pulsatility of LHRH secretion. The differences between time points were similar for the induced LH release. The results show that a short-day-like melatonin treatment caused a decrease in LH release in response to NMDA, as short days do. Furthermore, this modification in LH response to NMDA reflects a similar change in LHRH release. NTRODUCTON Melatonin regulates seasonal reproduction through changes in the pulsatile secretion of LHRH and LH [, 2]. This action of melatonin on the activity of LHRH neurons appears to be indirect and may involve several neuronal systems [3]. The results of several experiments suggest that inhibitory pathways, such as catecholaminergic ones, are responsible for a reduction in LHRH and LH pulse frequency during the anestrous season [4-7] and that a diminution of the activity of dopaminergic neurons at the level of the median eminence is associated with the reestablishment of a high LH pulse frequency by short days [8, 9]. A complementary hypothesis is that the increased LHRH secretion during the induction of breeding season also implicates stimulatory mechanisms. n support of this hypothesis, administration of N-methyl-D,L-aspartic acid (NMDA), an agonist of neuroexcitatory amino acid receptors, acutely stimulates LH secretion in seasonal species such as the hamster and the sheep [0-5]. However, this stimulatory effect is larger during periods of photoinhibited LH secretion than during photostimulated periods, suggesting that excitatory amino acids are involved in the photoperiodic regulation of LH secretion. The purpose of the present study was to determine whether NMDA-stimulated LH secretion Accepted January 6, 995. Received October 4, 994. 'This work was supported in part by a grant from "R6gion Centre." C.V. was supported by a Ph.D. grant from "R6gion Centre." ZCorrespondence. FAX: Present address: Reproductive Sciences Program, University of Michigan, 300 North ngalls Building, Ann Arbor, M is altered by melatonin-induced neuroendocrine activity in long-day-maintained ewes and whether this effect is mediated by hypothalamic LHRH release. Drugs MATRALS AND MTHODS NMDA, comprising a mixture of D and L isomers, was obtained from Sigma (Saint Quentin Fallavier, France). t was dissolved in 0.9% sterile and apyrogenic saline at room temperature just before injection. The total amount necessary for the ewes studied on a given day was dissolved in the same volume so that the concentration of the injected solution was constant, but the volume was adapted to the individual body weights. NMDA was injected intravenously. Preliminary xperiment: NMDA Dose Response Curve A dose response curve for the effect of NMDA on LH secretion was constructed in a preliminary experiment in order to test the effect of this substance on LH secretion in the ewe under our experimental conditions and to determine the dosage to use in the main experiment. This preliminary experiment was performed on four ovariectomized ewes treated with a 2-cm s.c. estradiol implant [6]. LH secretion in these animals was inhibited by exposure to long days (6L:8D) for 70 days. Three dosages were tested at 48-h intervals on the same animals (, 2, and 5 mg/kg BW) in an increasing dosage order. LH secretion was assessed in serial samples of jugular blood obtained 56

2 MLATONN AND LHRH RSPONS TO NMDA 57 every 20 min during the 2 h preceding the injection and every 0 min for 2 h thereafter. Main xperiment: ffects of NMDA on LHRH and LH Secretion at Different Periods of the Activation of LH Secretion by Melatonin The experiment was performed on adult ovariectomized le de France ewes, each bearing a 2-cm s.c. implant of estradiol and kept under long days (6L:8D) until the end of the experiment. Twenty-one animals were cannulated to access the hypothalamo-hypophysial portal system. All of these received s.c. melatonin implants (Melovine; generously provided by Hoechst, Hauxton, UK) on Day 0 of the experiment. The animals were allocated to one of three groups (groups, 2, 3 in companion to this paper [2]) according to the duration of melatonin treatment at the time of portal blood sampling. The effect of NMDA on LHRH and LH secretion was assessed at three different stages (one group per stage) according to the expected changes in LH secretion in response to the melatonin treatment. The first group (n = 6) was tested during the inhibition of LH secretion by long days, on Day -. The second group (n = 9) was tested just prior to the onset of the LH increase, on Day 39. The third group (n = 6) was tested when LH secretion was maximal, on Day 74. On each occasion, NMDA was administered by a venous catheter at a dose of 5 mg/kg BW. After the injection, portal and jugular blood was continuously sampled and fractions were collected every 0 min for 6 h to determine the effect of NMDA on LHRH and LH pulsatile secretion. Prior to this treatment, secretion of LHRH and LH had been studied for 7.5 (groups and 2) or 0.5 h (group 3) with an injection of LHRH.5 h before injection of NMDA [2]. The concentrations of LHRH in portal samples and of LH in jugular samples were analyzed by RAs as described previously [2]. Analysis of Data Preliminary experiment. The dose response curve was analyzed by a 3-factor (dosage, period relative to injection, time within period) repeated measures ANOVA on seven LH concentrations measured before and after the injection. Main experiment. The effects of NMDA on LHRH and LH secretion were analyzed by a 2-factor (between: group; within: time after injection) repeated measures ANOVA after logarithmic transformation. The responses were characterized by duration, amplitude, and the area under the curves. The duration was the time that elapsed between the injection and the first sample having a hormonal concentration as low as or lower than the mean concentration during the 6 h of spontaneous secretion preceding this experiment. n addition, in order that fluctuation of the basal level of LH not be included in the induced rise, the LH response was considered to have finished as soon as the -J u min FG.. NMDA dose response curve: mean + SM plasma LH concentrations before and after a single i.v. injection of NMDA. Arrow indicates the time of injection. Three dosages were tested 48 h apart on four ovariectomized and estradiol-treated ewes during inhibition of LH secretion induced by long days (6L:8D). Jugular blood samples were obtained every 20 min for 2 h before injection and every 0 min for 2 h thereafter. concentration fell lower than ng/ml. The amplitude was equal to the maximal value of the rise minus the mean concentration during the 6 h of spontaneous secretion preceding this experiment. The area under the curve was determined by multiplying the mean relative level during the rise by the duration of the rise. The mean relative level of the rise was defined as the mean concentration throughout the duration of the stimulation less the mean concentration during the first 6 h of spontaneous secretion. These three criteria were analyzed, after logarithmic transformation, by a -factor ANOVA, followed by a Fisher t-test to determine differences between groups. NMDA Dose Response Curve RSULTS.., njection of NMDA caused an acute increase in LH secretion characterized by a sustained and prolonged rise in plasma LH levels (Fig. ). This response was dose-dependent (Fig. ;p = 0.002) with mean LH levels peaking at.0, 2.0, and 2.8 ng/ml for the dosages of, 2, and 5 mg/kg BW, respectively. After injection of the highest dosage (5 mg/kg BW), LH secretion remained above baseline for more than 2 h. ffects of NMDA on LHRH and LH Secretion at Different Periods of the Activation of LH Secretion by Melatonin n the three situations studied, NMDA (5 mg/kg) caused an increase in LH secretion resulting from a simultaneous release of LHRH. However, the characteristics of this stimulation of both LHRH and LH secretion varied markedly among groups (Figs. 2 and 3; interaction between group and time after injection, p < 0.00 for both LHRH and LH levels). On Day -, in all animals, administration of NMDA caused an increase in LHRH secretion within 0-20 min. This in-

3 ...4~RP\ 58 VGU T AL. D- (N=6) 00 D l Lo _ g D+39 (N=9) - 00 D+39 r_ 0. m _ 0 = -J or 2.0 X.0 au 0.5 L "'i" alr CL c 0 -SC r -J CU co L J Cu D (N=6) A... min FG. 2. Mean (-+ SM) LHRH (top profiles) and LH (bottom profiles) concentrations for 6 h after an i.v. injection of 5 mg/kg of NMDA at three different times relative to the insertion of melatonin implants on Day 0, in ovariectomized and estradiol-treated ewes kept under long days. The bars represent the values of LHRH and LH concentrations during the first 6 h of spontaneous secretion preceding the injection of NMDA. LHRH and LH concentrations were calculated after logarithmic transformation of the data and are plotted on a logarithmic scale. N indicates the number of animals per group. r D+74 WKPZ min FG. 3. xamples of individual LHRH (top profiles) and LH (bottom profiles) secretory profiles for 6 h of spontaneous secretion and 6 h after an i.v. injection of 5 mg/kg of NMDA (shaded area) at three different times relative to the insertion of melatonin implants on Day 0. Portal and jugular blood samples were obtained every 0 min. LHRH and LH concentration values are plotted on a logarithmic scale. crease was much larger than a physiological pulse. ndeed, the amplitude of the peak varied between 25 and 38 pg/ ml, and LHRH levels remained elevated for 70 to 290 min (Figs. 2-4). Although LHRH levels remained elevated for a long time, they displayed fluctuations (Fig. 3). The stimulation by NMDA also resulted in a large (LH amplitude ranging from.8 to 0.4 ng/ml) and prolonged (80-30 min above baseline) increase in LH levels. During this rise, levels fluctuated and decreased gradually to reach baseline levels (Figs. 2 and 3). On Day 39, the characteristics of the stimulation of LHRH and LH secretion by NMDA were not different from those on Day -, despite a tendency toward a decrease in the mean values of the three criteria. The amplitude of the LHRH release varied between 34 and 87 pg/ml, and the concentration remained elevated for min. LH levels reached ng/ml and remained elevated for min (Figs. 2-4). On Day 74, the administration of NMDA was followed by release of LHRH. However, this secretory episode did

4 MLATONN AND LHRH RSPONS TO NMDA 59 not appear different from physiological pulses. n particular, it was brief, with a single elevated value for three of the ewes and with three or fewer elevated values for the other three ewes. Peak LHRH levels varied from 0.6 to 44 pg/ml. The resulting LH increase was also smaller (amplitude from 0.4 to 6.0 ng/ml) and shorter in duration (less than 80 min). Following the increase in LHRH and LH secretion, an inhibition of LHRH and LH pulsatile secretion was observed for about 2.5 h before resumption of secretory profiles typical of the physiological situation (Fig. 3). The ANOVA revealed that duration and area under the curve of the LHRH and LH rises differed among groups. These were smaller on Day 74 than on Day - and Day 39 (Fig. 4). Similarly, the amplitude of the LHRH rise was not different between Day - and Day 39 and decreased on Day 74 (Fig. 4). The changes in the amplitude of the LH rise paralleled those of LHRH but did not reach significance (Fig. 4). DSCUSSON u, 00 This experiment demonstrates that the injection of a glutamatergic agonist, NMDA, to ovariectomized and estradioltreated ewes causes an acute release of LHRH and that this stimulation is much reduced in ewes in which LH secretion has been increased by treatment with a melatonin implant. These data are consistent with those showing different degrees of stimulation of LH secretion by NMDA according to the photoperiodic situation. n the ram [2] and the hamster [3], NMDA administration resulted in increased LH secretion regardless of the physiological state, but this stimulation was larger in photoinhibited than in photostimulated animals. Our experiment extends these previous data in two directions. First, our data suggest that these modifications in the response of LH to NMDA according to the physiological state arise from modifications in LHRH response and do not reflect a change in pituitary responsiveness to LHRH. Secondly, this change in the sensitivity to NMDA was induced by a melatonin treatment in our experiment, not by photoperiod as in previous studies. Thus, our experiment shows that excitatory amino acids could be implicated in photoperiodic regulation of LHRH secretion independently of their probable functions as mediators of the effects of light on generating the daily melatonin pattern [7-9]. The main conclusion of this experiment, that the LHRH system is more sensitive to NMDA when LH secretion is low (i.e., before and during the 50-day latency after the beginning of melatonin treatment), may be explained by two hypotheses: first, that the phenomenon is simply a consequence of the degree of activity of the system and, secondly, that it is a mechanistic step of the control of LHRH pulsatility by melatonin. n favor of the first hypothesis, the modification of LHRH sensitivity to NMDA could reflect a decrease in the releasable stores of LHRH available at the level of the median eminence when the system is activated. nm_ ** m. 0 M LHRH rzlh._ -J -O3 FL U) D- D+39 D O J 0 X FG. 4. Mean ( SM) duration (top), area under the curve (middle), and amplitude (bottom) of the stimulation of LHRH (solid bars) and LH (hatched bars) secretion by NMDA (5 mg/kg) at three different times relative to the insertion of melatonin implants on Day 0. wes were ovariectomized, treated with estradiol, and kept under long days. Values were calculated after logarithmic transformation and are plotted on a logarithmic scale. deed, LHRH hypothalamic content is low when LHRH and LH secretion are maximal [20-22], and this is when NMDA stimulation is lowest. The temporary absence of LHRH and then LH pulses (or the decrease in pulsatility) after NMDA injection, observed in the third group, is also consistent with this depletion hypothesis. Moreover, it has been shown that some LHRH metabolites can inhibit the NMDA-induced LHRH release of hypothalamic explants in vitro [23]. The low response to NMDA in ewes with maximal LH secretion could be the consequence of an increased accumulation of LHRH metabolites in this situation of high LHRH release. Thus, available stocks of LHRH and the accumulation of LHRH metabolites could both explain the decrease in LHRH sys- v cc L

5 60 VGU T AL. tern sensitivity according to its level of activity. n our experiment, it is interesting to note that LHRH sensitivity to NMDA was the greatest when estradiol negative feedback on LHRH secretion was most important (i.e., Day - and Day 39). n addition, it seems that in some physiological situations sex steroids can modify excitatory amino acid mediation by changing either NMDA receptor density [24] or glutamate and aspartate content [25]. Furthermore, estradiol seems necessary in order to obtain a clear stimulatory effect of NMDA on LH secretion in adult animals [0, 26,27]. The lack of sensitivity to NMDA during the breeding season could therefore reflect a lack of stimulation of excitatory amino acid mediation by estradiol, since sensitivity to estradiol is lowest at this time. On the other hand, excitatory amino acids could be implicated in the regulation of LHRH secretion by melatonin. ndeed, short days or melatonin treatment could decrease the sensitivity of the LHRH system to NMDA. n agreement with the hypothesis of a possible implication of excitatory amino acids, it has been shown in the hamster that photoperiod can modify the density of the NMDA receptors in the preoptic area [28]. Another interesting observation from our experiment relates to the pattern of LHRH and LH secretion over a prolonged period (6 h) after a single administration of NMDA. Measurement of the secretion of LHRH, which is eliminated immediately from the hypothalamo-hypophysial portal blood, allows one to determine the duration of the secretion. n contrast, the LH levels reflect both the secretion and the kinetics of elimination. Our data demonstrate that on Day - and Day 39, NMDA caused a prolonged and sustained secretion of LHRH, very different from that observed during a normal physiological pulse. Furthermore, although it was not possible to detect pulses of secretion under the sampling conditions, the levels of LHRH, and of LH, fluctuated after NMDA administration. This could result from continuous secretion at a nonconstant rate produced by asynchronous random firing of LHRH neurons. Alternatively, it could suggest the existence of an episodic secretion with a frequency so high that hormonal concentrations did not remain basal for more than 0 min (sampling interval). We cannot, therefore, totally exclude the possibility that the induction of a prolonged secretion of LH and LHRH by a single bolus of NMDA constitutes a reactivation of the pulsegenerating system as suggested by Lincoln and Wu [2] in the ram. t is also interesting to note that the amplitude of the LHRH release in response to NMDA in ewes with low levels of LH secretion appears to be similar to that found during the preovulatory surge (about 00 pg/ml in our study; pg/ml during the surge [29]) and to be higher than that of physiological pulses (0-30 pg/ml [29]). t is clear that NMDA modifies LH secretion by modification of LHRH secretion [30-32]. However, it is unclear whether or not NMDA acts directly on LHRH neurons. Since NMDA, a polar molecule, is unable to penetrate the bloodbrain barrier rapidly, the median eminence, which is not protected by this barrier, could be a likely site for its action. n agreement with this hypothesis, it has been shown in monkeys that there is a close association between beaded LHRH immunoresponsive axons and glutamate immunoresponsive processes at the level of the stalk median eminence and particularly into its external layer and neurohemal contact zone [33]. However, a direct action of NMDA on LHRH cells is contested [34]: administration of NMDA to rats failed to induce c-fos expression, a marker of neuronal activation, in LHRH neurons [35, 36]. NMDA could act on LHRH secretion through neural systems such as catecholamines [35] or neuropeptides [37], which modulate the secretion of LHRH. n conclusion, our experiment shows that the secretion of LHRH induced by an injection of NMDA is lower in ewes in which LH secretion is stimulated by a melatonin treatment mimicking short days than in ewes in which LH secretion is inhibited. This suggests that endogenous excitatory amino acids might be involved in the action of melatonin on the seasonal control of reproduction. ACKNOWLDGMNTS We wish to thank Drs. M. Caldani, P. Chemineau, FJ. Karsch, and D.C. Skinner for help in the design of the study and comments on the manuscript; B. Delaleu, C. Fagu, and F. Maurice for their technical help; Mr. G. Durand and F. Paulmier and their team for assistance in the animal experimentation; and Mrs. D. Nowak for revision of the nglish manuscript. RFRNCS. Bittman L, Kaynard AH, Olster DH, Robinson J, Yellon SM, Karsch FJ. 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