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Department of Cell Biology and Physiology (S.R., M.J.D., T.M.P.), University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Harvard Reproductive Endocrine Sciences Center, Reproductive Endocrine Unit of the Department of Medicine (S.B.S., W.F.C.), Massachusetts General Hospital, Boston, Massachusetts 02114; and Department of Physical Therapy (C.R.P.), School of Health Sciences, Duquesne University, Pittsburgh, Pennsylvania 15282
Address all correspondence and requests for reprints to: Prof. Tony M. Plant, Ph.D., Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, S828A Scaife Hall, 3550 Terrace Street, Pittsburgh, Pennsylvania 15261. E-mail: plant1{at}pitt.edu.
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Introduction |
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We have previously shown that a continuous iv infusion of human metastin 45–54 (hu metastin 45–54) desensitizes GPR54-induced GnRH release as monitored indirectly by LH secretion in GnRH-primed agonadal juvenile monkeys (10). To use pituitary LH secretion as a sensitive bioassay for endogenous GnRH release in the juvenile monkey, the gonadotrophs must first be primed by a pulsatile infusion of exogenous GnRH (11) because the gonadotropin responsiveness of the pituitary at this stage of development is otherwise minimal (12). In this model, a continuous iv infusion of hu metastin 45–54 at 100 µg/h for 4 d elicited an initial and marked discharge of LH with a duration of approximately 3 h. This stimulatory phase was then followed by a precipitous drop in circulating LH concentrations despite continuous exposure of GPR54 to hu metastin 45–54. Interrogation of each component of the metastin-GPR54-GnRH neuron-GnRH receptor cascade with boluses of hu metastin 45–54, N-methyl-D-L-aspartic acid (NMDA), and GnRH, respectively, during the last 3 h of the continuous hu metastin 45–54 infusion indicated that the lesion in this pathway was restricted to desensitization of GPR54 (10).
Because of the potential clinical application of the latter finding, we sought to extend this observation to the fully adult, gonad-intact male rhesus monkeys exhibiting spontaneous endocrine activity in the hypothalamic-pituitary-testicular axis. On a body weight basis, the initial dose selected for the present study (200 µg hu metastin 45–54/h) was approximately equivalent to that employed in the previous experiments with the juvenile monkey. Some of the effects of this initial dose were equivocal, however, and therefore a higher dose (400 µg hu metastin 45–54/h) was also examined.
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Materials and Methods |
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Reagents
Hu metastin 45–54, equivalent to hu kisspeptin 112–121, was synthesized at the Peptide/Protein Core Facility of the Massachusetts General Hospital. A stock solution of the peptide (500 µg/ml) was prepared in 5–7% dimethylsulfoxide (DMSO) (Sigma Chemical Co., St Louis, MO) in sterile physiological saline (0.9% NaCl) (Abbott Laboratories, Chicago, IL) and stored at –80 C. For continuous iv hu metastin 45–54 infusion, the infusate (100 or 200 µg hu metastin 45–54/ml) was prepared in sterile Dulbecco’s PBS (DPBS without CaCl2 and MgSO4) (Life Technologies, Inc., Grand Island, NY) or sterile physiological saline and stored at 4 C. A 5–7% DMSO solution in sterile physiological saline was also prepared and stored at –80 C. This solution was diluted with sterile DPBS or physiological saline to prepare vehicle infusates with DMSO concentrations (1–2.8% DMSO) that matched those of the hu metastin 45–54 infusates. The vehicle infusates were also stored at 4 C. During continuous infusions of vehicle or peptide, a calibrated reservoir (Buretrol; Baxter Healthcare Corp., Deerfield, IL) containing the infusate was maintained at room temperature and refilled as required.
For bolus iv injection of hu metastin 45–54, 10- or 30-µg doses (in 1 ml sterile DPBS or physiological saline) were used. NMDA (Sigma-Aldrich Inc., St. Louis, MO) was dissolved in sterile physiological saline at a stock concentration of 100–200 mg/ml, and for bolus iv injection, a dose of 10 mg/kg body weight was prepared in 1 ml sterile saline and passed through a 0.22-µm filter (Fisher Scientific, Pittsburgh, PA) before administration. GnRH, synthesized at the Salk Institute (Contract N01-HD-0-2906), was obtained from Dr. A. F. Parlow, National Hormone and Peptide Program, Harbor-UCLA Research and Educational Institute, Los Angeles, CA. A stock GnRH solution was prepared at 1 mg/ml in sterile saline and stored at –20 C as previously described (13). For bolus iv injection, GnRH was diluted to 0.3 µg/ml in sterile saline and stored at 4 C.
Surgical procedures
The implantation of iv catheters (inner diameter, 0.040 in. and outer diameter, 0.085 in.; Stuart Bio-Sil, Sil-Med Corp., Taunton, MA) was performed under sterile conditions as described previously (11). Briefly, the animals were sedated with ketamine hydrochloride (10–20 mg/kg body weight, im) (Ketaject; Phoenix Scientific Inc., St. Joseph, MO) and anesthetized by isoflurane inhalation (1–2% in oxygen) (Abbott Animal House, North Chicago, IL). Two indwelling catheters, one placed in an internal jugular vein and the other in a femoral vein, were employed. One iv line was dedicated for continuous iv infusion of hu metastin 45–54 or vehicle and the other for blood sampling. Generally, the femoral line was used for infusions, but on occasion, patency was lost in the sampling line (jugular), and at those times the lines were switched so that the experiments could be maintained without additional surgery. The animals received a single im injection of penicillin (Pen-G, 40,000 U/kg body weight) (Phoenix Scientific) on the day of surgery. Postsurgically, the animals received twice-daily iv injections of a broad-spectrum antibiotic (Kefzol, 25 mg/kg body weight) (Apothecon, Princeton, NJ) and an analgesic (Ketofen, 2 mg/kg body weight) (Fort Dodge Animal Health, Fort Dodge, IA) for 4 d.
Collection of blood samples
Blood samples (1–3 ml) were withdrawn via the sampling iv catheter into heparinized syringes and transferred to sterile tubes, and the plasma was harvested after centrifugation. During periods of sequential sampling, packed blood cells were resuspended with sterile saline and returned to the respective animal. Plasma was stored at –20 C until required for assays.
Experimental design
Effect of a 200 µg/h continuous iv infusion of hu metastin 45–54 on circulating LH and testosterone (T) concentrations.
This experiment was conducted in four monkeys 1–4 wk after catheterization. Initially, the animals were subjected to a frequent blood sampling protocol between 1600–2200 h (every 20 min) to establish pretreatment evening patterns of pulsatile LH and T secretion. The hypothalamic-pituitary-Leydig cell axis of male monkeys under the present laboratory conditions is diurnally modulated with maximal activity occurring between 1700 and 0600 h (14). A 98-h continuous iv hu metastin 45–54 or vehicle infusion was initiated a few days after the collection of sequential blood samples. On the first day of infusion (d 1), a single bolus iv injection of hu metastin 45–54 (10 µg in 1 ml) was given at 0900 h, and a continuous hu metastin 45–54 (n = 2) or vehicle (n = 2) infusion was initiated 1 h later. On d 3 of continuous hu metastin 45–54 or vehicle infusion, moment-to-moment changes in circulating concentrations of LH and T were again monitored from 1600–2200 h. On d 4, during the last 3 h (0900–1200 h) of continuous hu metastin 45–54 or vehicle infusion, the animals received in sequence and at hourly intervals single bolus iv injections of 10 µg hu metastin 45–54, NMDA (10 mg/kg body weight), and 0.3 µg GnRH, at the end of which the continuous hu metastin 45–54 or vehicle infusion was terminated at 1200 h. Although it is recognized that the invariant order of the foregoing challenges may have determined, in part, the relative responses, in the confines of the present experimental design, the response to bolus administration of metastin 45–54 was of utmost importance, and therefore this peptide was always injected first. One day later (d 5), the animals received another iv bolus of 10 µg hu metastin 45–54 at 0900 h.
The following additional blood samples were collected: 1) on d 1 at 10 min before and at 10, 20, 30, and 50 min after the bolus iv injection of hu metastin 45–54 and during the first 12 h of continuous hu metastin 45–54 or vehicle infusion at 10, 20, 30, 50, 70, 90, 110, 130, 150, 170, 360, and 720 min; 2) on d 2 and 3, a single blood sample collected in the morning and evening, at approximately 1000 and 2200 h; 3) on d 4, a morning sample collected at approximately 1000 h and a series of blood samples collected 10 min before and at 10, 20, 30, and 50 min after each of the hu metastin 45–54, NMDA, and GnRH challenges; and 4) on d 5, a series of blood samples collected 10 min before and at 10, 20, 30, and 50 min after the bolus of hu metastin 45–54.
Moment-to-moment changes in circulating concentrations of LH and T were again monitored between 1600–2200 h 4–12 d after termination of the continuous infusions. After an interval of a week or more, the experimental protocol was repeated, adopting a crossover design. It should be noted that, in most instances, the evening window of frequent blood sampling (1600–2200 h) at the end of one leg of the experiment also served as the pretreatment pulse bleed for the crossover design.
Additionally, nonheparinized blood samples were collected before, during, and immediately after termination of the continuous hu metastin 45–54 infusions with the aim of measuring circulating hu metastin 45–54 concentrations at a later date.
Effect of a 400 µg/h continuous iv infusion of hu metastin 45–54 on circulating LH and T concentrations.
The effects of a 400 µg/h continuous iv hu metastin 45–54 were studied in five adult monkeys using a protocol very similar to that described above. Three of the monkeys that received the high-dose infusion had earlier received the 200 µg/h dose during the first experiment, and in these animals, the two experiments were separated by an interval of 2–6 months. Four of the five animals first received the continuous high-dose hu metastin 45–54 infusion followed by vehicle infusion. The iv hu metastin 45–54 bolus on d 4 and 5 was administered at 10 µg in two animals and at 30 µg in the remaining three monkeys. In one animal, the challenge dose of NMDA on d 4 of vehicle infusion was not administered.
Assays
Plasma LH levels were measured using a homologous (macaque) RIA as described previously (15). The minimal detectable concentration of the LH assay ranged from 0.12–0.32 ng/ml, and the mean intra- and interassay coefficients of variation were 9 and 16%, respectively.
Plasma T levels were determined using a commercially available solid-phase RIA kit (Total T; Diagnostic Products Corp., Los Angeles, CA), as described previously (16). The minimal detectable concentration of the T assay ranged from 0.01–0.06 ng/ml, and the mean intra- and interassay coefficients of variation were 6 and 10%, respectively.
Statistical analyses
The significance of differences in mean values of LH and T concentrations, LH pulse frequency, and LH pulse amplitude within and between treatments was determined by two-way ANOVA with repeated measures followed by Student-Newman-Keuls multiple range test. A mean LH and T concentration during each 6-h window (1600–2200 h) of frequent blood sampling was obtained for each monkey by averaging the individual values. Hormone levels below the sensitivity of the assay were assigned a concentration equivalent to the minimum detectable concentration in the respective LH assay.
Episodes of LH secretion (pulses) during each of the 6-h windows (1600–2200 h) of sequential sampling were identified by the Pulsar pulse detection algorithm (17) that determines the number and amplitude of hormone pulses. The G values used, which produce a 1% false-positive rate, were: G(1) = 4.40, G(2) = 2.60, G(3) = 1.96, G(4) = 1.46, and G(5) = 1.13.
The significance of differences in the magnitude of the LH response (LH concentration at 10 min after injection minus the preinjection value) to the bolus injections of metastin 45–54, NMDA, and GnRH was determined using paired t test.
All values are expressed mean ± SEM. Differences were considered significant at P < 0.05.
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Results |
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). Initiation of the continuous low- and high-dose infusions of hu metastin 45–54 1 h after the injection of a bolus of the peptide produced a second and more pronounced increment in circulating LH that reached peak levels of approximately 8 and 13 ng/ml 2 h into the low- and high-dose infusion, respectively (Fig. 1). Thereafter, however, circulating LH levels declined despite continuous metastin 45–54 administration, and by 24 h, they had reached or were approaching concentrations comparable with those observed before the administration of metastin 45–54 (Fig. 1). Vehicle infusions did not stimulate LH secretion (Fig. 1).
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). However, the pronounced decline in circulating LH concentrations that was observed from about noon on d 1 after initial stimulation of this gonadotropin by both the low- and high-dose infusions was not associated with a corresponding fall in circulating T concentrations (Fig. 1). The maintenance of T secretion throughout d 1 in the face of declining LH levels was particularly marked at the high-dose metastin 45–54 infusion; at the end of the first day of metastin 45–54 administration, the mean circulating T concentration was 18 ± 5 ng/ml, which compared with a value of 2 ± 1 ng/ml for vehicle (Fig. 1).
Moment-to-moment changes in circulating LH and T concentrations observed during the evening hours on d 3 of the continuous low-dose infusion of metastin 45–54 were indistinguishable from those during the corresponding sampling window of the vehicle infusion (data not shown). In marked contrast, the high-dose metastin 45–54 infusion was associated with a suppression of pulsatile LH levels during the evening window of sequential sampling on d 3 (Fig. 2), with the mean LH value of 0.4 ng/ml significantly (P < 0.05) lower than those before (1.4 ng/ml) and after (1.2 ng/ml) peptide treatment and that during d 3 of the corresponding vehicle infusion (1.4 ng/ml) (Fig. 3). The 70% reduction in circulating LH levels during the high-dose metastin 45–54 infusion was associated with a 40% decrease (P < 0.05) in mean plasma T concentration during the evening of d 3 (Fig. 4). When circulating T concentrations were expressed relative to the concentration of LH at the time of the steroid measurement (i.e. [T]:[LH]) in the morning and evening samples collected at approximately 1000 and 2200 h on d 2, 3, and 4, the [T]:[LH] ratios were invariably exaggerated during high-dose metastin 45–54 infusion compared with those during vehicle infusion (Table 1).
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and 4).
The mean number of LH pulses and the mean LH pulse amplitude observed during the evening windows of sequential sampling before, during (d 3), and after the low- and high-dose metastin 45–54 infusions are shown together with the corresponding mean LH concentrations in Fig. 3. In the case of the low-dose infusion of metastin 45–54, there were no significant differences for these parameters between peptide and vehicle treatments (Fig. 3). Approximately three LH pulses, with mean pulse amplitude of 1 ng/ml, were observed during the 6-h evening window of sampling before, during, and after low-dose metastin 45–54 or vehicle infusion (Fig. 3). In contrast, during the high-dose metastin 45–54 infusion, a decrease in both LH pulse frequency (four pulses per 6 h to less than one pulse per 6 h, P < 0.05) and LH pulse amplitude (2 ng/ml to 0.5 ng/ml, P < 0.05) was observed during the evening window of sequential sampling on d 3 (Fig. 3).
The effects on LH release of single, sequential bolus iv injections of metastin 45–54, NMDA, and GnRH on d 4 during the last 3 h of continuous low- and high-dose metastin infusion and the corresponding effects on LH release during vehicle infusion are shown in Fig. 5. Although the magnitude of the LH response evoked by the NMDA and GnRH challenges during the continuous infusion of the low-dose metastin 45–54 infusion were similar to those observed during vehicle infusion, the overall LH releasing action of the bolus metastin injection was compromised (Fig. 5). The response was abolished in two animals and truncated in two. In all four monkeys, the ability of the bolus metastin 45–54 challenges to elicit LH release was fully restored 24 h after termination of the continuous low-dose infusion (Fig. 5). Interrogation with the same challenges during the high-dose metastin 45–54 infusion indicated a more pronounced and prolonged suppression of the axis (Fig. 5). At this dose, the LH-releasing action of the bolus metastin challenge on d 4 during the last 3 h of continuous infusion was abolished even though the bolus dose of the peptide was increased to 30 µg in three of the five animals studied. Moreover, the LH responses to NMDA and GnRH were also compromised during continuous infusion of high-dose metastin 45–54. Finally, in contrast to low-dose metastin 45–54 infusion, only a partial recovery in LH response to the metastin 45–54 bolus was noted 24 h after termination of the peptide infusion at the high dose (Fig. 5).
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Discussion |
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Although the failure of continuous administration of kisspeptins to sustain LH secretion at elevated levels has now been reported in monkey, rat, and sheep, continuous intracerebroventricular mouse metastin 43–52 administration (0.25 nmol/h per animal) to male hamsters maintained under photoperiodic conditions (short days) that induce testicular regression resulted in a resurgence of spermatogenic activity (20). In the latter study, neither LH nor FSH levels were monitored, but the suppression of testicular function induced by exposure to short days was presumably reversed by metastin 43–52-induced gonadotropin secretion, although a direct testicular action, albeit unlikely because of the dose and route of administration, should not be completely excluded (see below).
In both the earlier study of the agonadal juvenile male monkey (10) and the present study of the testes-intact adult monkey, circulating LH concentrations declined after the surge in LH secretion during the initiation of continuous metastin 45–54 administration to reach values indistinguishable from those observed during vehicle infusion by the next day. As in the agonadal juvenile monkey, the failure of continuous administration of metastin 45–54 to sustain LH secretion at elevated levels in the intact adult monkey was due primarily to desensitization of GPR54. This was reflected in the finding that the LH discharge induced by bolus administration of metastin 45–54 was truncated during the low-dose peptide infusion and abolished at the high-dose infusion, as it was in the study with agonadal juveniles (10). In contrast to the earlier study of juvenile males, in which the failure of continuous metastin 45–54 to sustain LH secretion could be accounted for solely by down-regulation of GPR54 signaling, the high-dose metastin 45–54 infusion to intact adult males led to a reduction in the magnitude of the LH discharge induced by the NMDA and GnRH challenge, although that for the glutamate challenge was not statistically significant, indicating that pituitary responsiveness to GnRH was compromised. Pituitary expression of GPR54 has been reported in the rodent and human (5, 21), but to date, reports of the action of kisspeptins directly at the pituitary level have been inconsistent (22). That down-regulation of the hypothalamic-pituitary axis of the adult male by the high-dose infusion of metastin 45–54 was more severe than that reported earlier for the agonadal juvenile receiving a similar dose of peptide (40 µg/kg vs. 33 µg/kg body weight, adult vs. juvenile, respectively) is further indicated by the finding that the ability of the metastin 45–54 bolus to elicit an LH discharge was not fully restored 24 h after termination of the continuous high-dose metastin infusion.
Desensitization of GPR54 by the high-dose continuous infusion of metastin 45–54 was associated with a marked suppression in spontaneous LH secretion that was occasioned by both a deceleration in LH pulse frequency and a reduction in LH pulse amplitude. Because a reduction in LH pulse amplitude may result in LH increments falling below threshold for detection as an LH pulse, it remains to be confirmed whether the decrease in LH pulse frequency, which generally reflects GnRH pulse frequency (23), was due to a metastin 45–54-induced retardation of the GnRH pulse generator. Whatever the case may be, it is nevertheless reasonable to conclude that the decrease in spontaneous LH secretion during high-dose metastin 45–54 administration was the result of reducing excitatory kisspeptin inputs to the GnRH neuronal network as a result of GPR54 desensitization.
Although the high-dose continuous metastin 45–54 infusion in the adult monkey abolished the LH-releasing action of the exogenous bolus of ligand, a component of spontaneous LH pulsatility was preserved. This result is consistent with the earlier finding of spontaneous nocturnal elevations in LH release in agonadal juvenile males receiving a similar continuous infusion of metastin (10). At the time of the earlier study, we concluded that spontaneous activity in this neuroendocrine axis in the face of GPR54 desensitization induced by continuous exposure to iv administered metastin 45–54 was due to one of two possibilities. First, the dose of metastin 45–54 employed may not have been sufficient to achieve desensitization of GPR54 in all hypothalamic areas due, perhaps, to incomplete penetration across the blood-brain barrier. Second, the component of LH secretion that is preserved in the presence of continuous exposure to metastin 45–54 may be driven by pathways that do not involve kisspeptin-GPR54 signaling. These same possibilities may be put forward to account for the failure in the present study of the continuous high-dose infusion of metastin 45–54 to completely abolish pulsatile LH secretion in the adult male.
Although the experimental design of the present study was not crafted to examine potential direct effects of metastin on testicular function systematically, changes in the quantitative relationship between circulating LH concentrations and T levels at various times during the continuous infusion of metastin 45–54 indicate that kisspeptins may amplify LH-induced secretion of this androgen by the monkey testis. Specifically, plasma T concentrations for a given LH stimulus (i.e. plasma [T]:plasma [LH]) were invariably greater during high-dose metastin 45–54 infusion than during vehicle infusion. In contrast to the foregoing findings indicating that kisspeptins may potentiate LH-induced T secretion by the monkey Leydig cell, in adult male rats 13 d of continuous treatment with 8.3 nmol/kg·h of full-length metastin resulted in a significant LH-independent reduction in circulating T levels that was associated with decreased testicular weight and degeneration of the seminiferous epithelium (19). Although a molecular basis for a direct kisspeptin action in the testis is extant because GPR54 expression has been noted in the testis (3, 5, 21), the intratesticular function of kisspeptin signaling and the identity of the cells involved requires systematic study.
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Acknowledgments |
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Footnotes |
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A preliminary report of this work was presented at the Sixth International Congress of Neuroendocrinology, Pittsburgh, June 2006, Abstract P243.
Disclosure Statement: The authors have nothing to disclose.
First Published Online April 5, 2007
Abbreviations: DMSO, Dimethylsulfoxide; DPBS, Dulbecco’s PBS; GPR54, G protein-coupled receptor 54; hu, human; NMDA, N-methyl-D-L-aspartic acid; T, testosterone.
Received February 12, 2007.
Accepted for publication March 21, 2007.
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