We have previously shown that a decrease in γ-aminobutyric acid (GABA) tone and a subsequent increase in glutamatergic tone occur in association with the pubertal increase in luteinizing hormone releasing hormone (LHRH) release in primates. To further determine the causal relationship between developmental changes in GABA and glutamate levels and the pubertal increase in LHRH release, we examined monkeys with precocious puberty induced by lesions in the posterior hypothalamus (PH). Six prepubertal female rhesus monkeys (17.4 ± 0.1 mo of age) received lesions in the PH, three prepubertal females (17.5 ± 0.1 mo) received sham lesions, and two females received no treatments. LHRH, GABA, and glutamate levels in the stalk-median eminence before and after lesions were assessed over two 6-h periods (0600–1200 and 1800–2400) using push-pull perfusion. Monkeys with PH lesions exhibited external signs of precocious puberty, including significantly earlier menarche in PH lesion animals (18.8 ± 0.2 mo) than in sham/controls (25.5 ± 0.9 mo, P < 0.001). Moreover, PH lesion animals had elevated LHRH levels and higher evening glutamate levels after lesions, whereas LHRH changes did not occur in sham/controls until later. Changes in GABA release were not discernible, since evening GABA levels already deceased at 18–20 mo of age in both groups and morning levels remained at the prepubertal levels. The age of first ovulation in both groups did not differ. Collectively, PH lesions may not be a good tool to investigate the mechanism of puberty, and, taking into account the recent findings on the role of kisspeptins, the mechanism of the puberty onset in primates is more complex than we initially anticipated.
- timing of puberty
- luteinizing hormone releasing hormone
- lesions in the hypothalamus
the concept that an increase in pulsatile luteinizing hormone releasing hormone (LHRH) release triggers the onset of puberty has been well established (26, 44, 45). However, the mechanism of the pubertal increase in LHRH release remains unclear (39). It has been reported that children with tumors or hamartomas in the hypothalamus exhibit precocious puberty (37). A previous study from our laboratory showed that bilateral lesions made in the posterior hypothalamus of female rhesus monkeys during the prepubertal stage result in an early rise in luteinizing hormone (LH) release followed by precocious puberty (42). Moreover, similar lesions in ovariectomized prepubertal females result in early pubertal LH increases and accelerated timing of the estrogen positive feedback effect on the LH surge when compared with those in ovariectomized sham control females (35), indicating that the hypothalamic lesion-induced LH increase in prepubertal monkeys is independent of ovarian steroid hormones and is suggestive that lesions cause changes in the control mechanism of the LHRH neurosecretory system.
A series of studies in our laboratory indicate that an increase in LHRH release is associated with a decrease in γ-aminobutyric acid (GABA) followed by an increase in glutamate in the stalk-median eminence (S-ME) when female rhesus monkeys undergo normal puberty (21, 41). The importance of the decrease in GABA tone at the onset of puberty is further suggested by our observation that the GABAA receptor blocker, bicuculline, results in precocious puberty (15). However, it is still unclear whether the pubertal increase in LHRH release is causally related to the decrease in GABA release followed by an increase in glutamate release. Although producing lesions in the hypothalamus may not be the best approach, this is the only known method for induction of precocious puberty in nonhuman primates.
Therefore, the objective of this study is 1) to determine if precocious puberty induced by posterior hypothalamic lesions is the result of an increase in LHRH release in the S-ME and 2) to examine whether hypothalamic lesion-induced LHRH increases occur in association with a decrease in GABA release and an increase in glutamate release.
MATERIALS AND METHODS
Eleven female rhesus monkeys (Macaca mulatta), born at the Wisconsin National Primate Research Center, at 13–14 mo of age were used in this study. The animals were housed in pairs (172 × 86 × 86 cm) under controlled lighting conditions (lights on 0600–1800 and lights off 1800–0600) with room temperature maintained at 22°C. The monkeys were fed Purina Monkey Chow once every morning, supplemented with fresh fruit every afternoon. Tap water was available ad libitum. The protocol for this study was reviewed and approved by the Animal Care and Use Committee, University of Wisconsin, and all experiments were conducted under the guidelines established by the National Institutes of Health and United States Department of Agriculture.
Six of the 11 monkeys were assigned to the lesion group, whereas 3 of the 11 monkeys were assigned to the sham control, and the other 2 monkeys remained intact. Body weights were recorded weekly, and daily observations on development of sex, skin color, and menstruation were made throughout the entire experimental period, as described previously (9). Blood samples (3 ml) were collected one time per week for measurement of circulating hormones.
Experiments were designed to observe acute and chronic effects of hypothalamic lesions on precocious puberty. Lesions were made between 17.0 and 17.9 mo of age. Acute effects of posterior hypothalamic lesions on release of LHRH, GABA, and glutamate were to occur in the first 3 mo postoperatively (before 21 mo of age), whereas the chronic effects of posterior hypothalamic lesions on LHRH release would occur throughout the pubertal process.
At ∼14 mo of age, the monkeys were anesthetized with isoflurane, and cranial pedestals were implanted on their skulls using a stereotaxic apparatus. The center of the cranial pedestal was positioned above the third ventricle recess using x-ray ventriculograms, with which the third ventricle was visualized (11). The cranial pedestals allowed us to collect in vivo perfusates in the S-ME from monkeys at various ages during their development, as described below. To obtain control data in all animals, push-pull perfusion experiments were conducted 6 to 9 wk after the cranial pedestal implantation (between 15.5 and 17.0 mo of age), as described below. Between 17.0 and 17.9 mo of age, bilateral posterior hypothalamic lesions were made through cranial pedestals using x-ray ventriculography. The lesions were made by passing a radiofrequency current through a thermister electrode with a tip diameter of 0.7 mm, as described previously (35, 42). Briefly, the radiofrequency current was raised slowly over 30 s to bring the tip temperature to 73°C, which was maintained for 1 min, followed by a gradual current reduction during the 30-s period. In sham controls, the identical procedure was applied, except no radiofrequency current was passed through the electrode. Intact controls did not receive any experimental procedure. Subsequently, in all animals, except for one animal in the lesion group (see below), a push-pull perfusion experiment was conducted between 18 and 20 mo of age. In one lesion animal, only one push-pull experiment was conducted after lesions. To examine the chronic effect of lesions, push-pull perfusion experiments were conducted at 3- to 5-mo intervals until the animals ovulated for the first time.
To assess the changes in release of LHRH, GABA, and glutamate in the S-ME, the push-pull perfusion method was used. Before initiation of the push-pull perfusion experiments, the monkeys were adapted to the researchers and the experimental environments, as described previously. Before the perfusion experiment (3 days), the animals were anesthetized with ketamine and xylazine and placed in a stereotaxic apparatus. An outer cannula (20 gauge) with an inner stylet (28 gauge) was inserted in the median eminence-stalk using a hydraulic microdrive unit (model MO95-B; Narishige Instrument, Tokyo, Japan), which allows three-dimensional microadjustments for accurate placement. Cannula placement was visualized with x-ray ventriculograms and compared with the ventriculograms obtained during pedestal implantation. After implantation, the animals were placed in a primate chair and allowed 3 days to recover before the experiment started. On the day of the experiment, the stylet was replaced with an inner cannula (28 gauge), and artificial cerebrospinal fluid was infused through the inner (push) cannula, while the perfusate samples were collected from the outer (pull) cannula at 10-min intervals. Perfusate samples were collected for 6 h in the morning (0600–1200) and 6 h in the evening (1800–2400) using two peristaltic pumps with identical flow speeds (∼22 μl/min).The order of morning and evening sampling sessions was randomized. Perfusates were aliquoted to 150 μl for LHRH assay and the remaining (∼70 μl) for GABA and glutamate assay and stored at −70°C.
The LHRH levels in all perfusates were measured using RIA antiserum R1245, kindly provided by Dr. T. M. Nett (Colorado State University, Fort Collins, CO). The method for LHRH assay has been reported previously (11). Synthetic LHRH (Richelieu Biotechnologies, Montreal, Quebec, Canada) was used for the radiolabeled antigen and reference standard. The antigen-antibody complex was precipitated with a goat anti-rabbit gamma globulin. Sensitivity of the assay was 0.1 pg/tube. Intra- and interassay coefficients of variation were 8.1 and 11.3%, respectively.
The GABA and glutamate levels in one set of perfusate samples before and after lesions (or sham surgery in sham controls or at an equivalent age in intact controls) were measured by HPLC with electrochemical detection, as described previously (21, 41). Two aliquots from the same perfusate samples used for LHRH measurements, at 20 μl each (one for GABA and the other for glutamate), were derivatized with 45 μl of a mixture containing o-phthaldialdehyde reagent and 2-methyl-2-propanethiol (Sigma, St. Louis, MO) and separated by centrifugation. Samples were injected at 50 μl on a C18 column with a mobile phase consisting of 2.5 mM sodium phosphate dibasic, 0.5 mM EDTA, 40% methanol, and 10% acetonitrile at pH 7.1 for GABA assay or 5 mM sodium phosphate dibasic, 0.5 mM EDTA, 40% methanol, and 5% acetonitrile at pH 6.9 for glutamate assay. Minimum detectabilities of GABA and glutamate were 0.2 and 2.0 pg/20-μl sample, respectively.
For evidence of first and second ovulations, progesterone in serum samples was measured by RIA (40). The ages of first and second ovulations were assessed by the pattern of the sex-skin color breakdown followed by progesterone levels >1 ng/ml.
After their second ovulation, the lesion and sham animals were anesthetized and perfused with 4% buffered paraformaldehyde solution. The brains were removed and placed in the cryoprotectant sucrose PBS (30 g/100 ml). Serial sections were cut by cryostat in the frontal plane at 50 μm, and every 10th slide was stained with cresyl violet to visualize the region of the lesions using an atlas by Bleier (2) for reference. To identify the damage of the LHRH neuronal system by lesions, remaining serial sections were systematically immunostained with a cocktail of LR1 and GF-6 as described previously (30, 33), and tissue sections were further analyzed.
The data from the three sham lesion controls and two intact animals were pooled together as the control group (sham/controls, n = 5), since they were very similar. Because one of the six lesion animals had to be discontinued 4 wk after menarche at 20.5 mo of age, the lesion group became n = 5 after menarche. In one of the six lesion animals, GABA and glutamate were not measured. Acute lesion effects on LHRH, GABA, and glutamate levels were examined by repeated-measures ANOVA followed by post hoc Tukey's test using hourly mean from individual animals. Lesion effects on the pulsatility of LHRH release were assessed with the PULSAR computer algorithm (20). The key parameters for PULSAR were similar to those previously reported (49), i.e., the cutoff criteria for G1–G5 were 3.8, 2.6, 1.9, 1.5, and 1.2, respectively, and the intra-assay coefficients of variation for LHRH were described by the formula y = (3.38x + 3.14)/100. Chronic effects of posterior hypothalamic lesions on LHRH release were examined from the data obtained periodically after 21 mo of age through the confirmation of first ovulation: developmental changes in LHRH release at the age of 21–24 mo, 25–29 mo, 30–34 mo, and >35 mo in lesion and sham/control groups were compared using hourly mean from individual animals. Again, if multiple push-pull perfusion experiments were conducted in an animal during an age category, the results from only one experiment were used for the calculation. Chronic effects on GABA and glutamate levels were not assessed because of the large total sample number and the amount of labor required for GABA and glutamate analysis with HPLC in each sample. The effects of the treatments and ages on LHRH levels were examined by repeated-measures ANOVA followed by post hoc Tukey's test using hourly mean from individual animals. The effects of lesions on the age of menarche, first and second ovulations, and body weights at these events were examined with Student's t-test. Significance was attained at P < 0.05.
Posterior hypothalamic lesions on timing of puberty.
Posterior hypothalamic lesions resulted in precocious menarche (Table 1). The age (18.8 ± 0.2 mo) at menarche in the lesion group was significantly (P < 0.001) earlier than in the sham/control group (25.5 ± 0.9 mo). However, the ages of first and second ovulations in the lesion group (40.1 ± 3.4 and 44.9 ± 4.1 mo) were not significantly different from those in the sham group (41.3 ± 1.3 and 42.9 ± 1.3 mo).
The body weight (2.6 ± 0.1 kg) at menarche in the lesion group was also significantly (P < 0.01) smaller than that (3.2 ± 0.1 kg) in the sham/control group (Table 1). However, body weights at first and second ovulations (5.3 ± 0.5 and 5.5 ± 0.4 kg, respectively) were not different from those (5.2 ± 0.2 and 5.2 ± 0.2 kg) in the sham/control group.
Acute effects of posterior hypothalamic lesions on LHRH release.
The morning and evening mean LHRH levels before the lesion surgery in the lesion group and those in sham/controls at the equivalent age were low and similar between the two groups (Figs. 1 and 2). After posterior hypothalamic lesions, the morning and evening LHRH levels increased as shown in the example in Fig. 1. Similarly, mean LHRH levels (±SE) after lesions in the lesion group were significantly (P < 0.01) higher than those before lesions (Fig. 2). In contrast, the morning and evening LHRH levels within 3 mo in the sham/control group did not significantly change. The mean LHRH levels after the surgery in the lesion group were also higher (P < 0.01) than those in the sham/control group at the corresponding age (Fig. 2).
The results from PULSAR analysis indicated that the interpulse intervals in the morning and evening at 18–20 mo of age were significantly reduced after lesions compared with those before lesions at 15–17 mo of age (P < 0.01 for morning; P < 0.001 for evening; Table 2) as well as those in sham/controls at the corresponding 18–20 mo age (P < 0.01). Similarly, the pulse amplitudes in both morning and evening were significantly (P < 0.05) increased after lesions. Both interpulse intervals and pulse amplitude in sham/controls were not different between 15–17 and 18–20 mo of age (Table 2).
Acute effects of posterior hypothalamic lesions on GABA and glutamate levels.
Before hypothalamic lesions, the evening GABA levels in juvenile animals at 15–17 mo of age in lesion group were significantly higher (P < 0.01) than morning levels. After lesions, the evening GABA levels became significantly lower than those before lesions (P < 0.01; Fig. 3). The morning GABA levels were not affected by lesions. At 18–20 mo of age, evening GABA levels in lesion animals became lower than those in the morning (P < 0.05). In the sham/control group at 15–17 mo of age, the nocturnal increase was not significant. However, to our surprise, evening GABA levels in the sham/control group at 18–20 mo of age were significantly lower than those at 15–17 mo of age as well as those in the morning at the same age (for both P < 0.05; Fig. 3).
Before the treatment, there was no difference in morning and evening glutamate levels. After lesions, both morning and evening levels became significantly higher (P < 0.05 for AM and P < 0.01 for PM) than those before lesions (Fig. 4). The evening glutamate levels were also higher than those in sham/control at the corresponding age (P < 0.05; Fig. 4). At 18–20 mo of age, evening glutamate levels in the lesion group became higher than morning levels (P < 0.05). In contrast, neither evening nor morning glutamate levels in the sham/control group at 15–17 mo of age were different from those at 18–20 mo of age. Similarly, no circadian changes in glutamate levels in the sham/control group were observed at either age (Fig. 4).
Chronic effects of posterior hypothalamic lesions on LHRH release.
After menarche, both morning and evening LHRH levels in the lesion group further increased at 21–24 mo of age, and stayed at an elevated level until 30–34 mo of age (P < 0.001; Figs. 5 and 6). At the age before first ovulation (35+ mo), the evening LHRH levels in the lesion group further elevated, reaching the plateau value (Figs. 5 and 6). In contrast, both morning and evening LHRH levels in the sham/control group remained low through 24 mo of age, and evening LHRH levels at 25–29 mo finally became significantly higher (P < 0.05) than those at the juvenile age coinciding with menarche (Figs. 7 and 8). After 30 mo, both morning and evening LHRH levels in the sham/control group further increased, achieving the highest values, which were also highly significant (P < 0.001) compared with those at 15–17 mo of age. LHRH levels in the lesion group before 25 mo of age were also significantly higher than in the sham/control group (P < 0.001–0.01), but LHRH levels after 25 mo of age did not differ from those during the corresponding age in the sham/control group (Fig. 6). Evening LHRH levels in the lesion group were significantly higher than morning levels during all periods examined after lesions, whereas the evening LHRH levels were only significant after 25 mo of age in sham/control animals (Figs. 6 and 8).
The results from PULSAR analysis indicated that, in the lesion group, the interpulse intervals of LHRH release in both morning and evening after 21–24 mo of age were continuously shorter than those of the prelesion controls at 15–17 mo throughout the experiment (Table 2). Similarly, the pulse amplitudes in both morning and evening after 21–24 mo of age were also significantly higher than the prelesion controls at 15–17 mo throughout the experiment. However, comparison between the lesion and sham/control groups suggested that the morning interpulse intervals and both morning and evening pulse amplitudes in the lesion group were significantly different only at 21–24 mo of age, but not older, because in sham/control animals the interpulse interval and pulse amplitude both underwent pubertal changes (Table 2).
The lesioned areas of the hypothalamus in this study were similar (Fig. 9) but slightly more ventral to those observed in our previous study (42). The lesion scars were found bilaterally crossing the midline of the hypothalamus. The maximum extent of lesions was 3.1 ± 0.2 mm rostrocaudally. The areas included in all lesion animals were the premammillary area, a posterior portion of the infundibular (arcuate) nucleus, ventral portion of the ventromedial nucleus, the ventral portion of the dorsomedial nucleus, and anterior portion of the mammillary body. Although in one of the six monkeys the median eminence was slightly damaged, in the remaining five monkeys the median eminence was completely spared. In four of the six monkeys, only the caudal portion of the infundibular nucleus was lesioned, and in the two of the six monkeys ∼40% the infundibular nucleus was lesioned. Immunocytochemical staining indicated that LHRH fibers and perikarya in the stalk region were abundant in the lesion animals, but there were no LHRH fibers and perikarya in the lesioned area.
The results of the present study indicate that an increase in LHRH release occurs when the age of puberty was advanced by lesions in the posterior hypothalamus. Although it has been clearly shown that pulsatile LHRH release increases at the onset of spontaneous puberty in monkeys and rats (38, 44), to date an elevated release of LHRH in association with precocious puberty has only been shown indirectly by measurement of LH. Clinical studies have demonstrated that pulsatile LH release increases in association with precocious puberty in humans and that injection of an LHRH superagonist arrests precocious puberty, presumably because of pituitary downregulation (see Refs. 3 and 34). In rhesus monkeys, an increase in LH release occurs when precocious puberty is induced by posterior hypothalamic lesions (42), infusion of the GABAA receptor antagonist bicuculline in the basal hypothalamus (15), systemic injection of insulin-like growth factor I (IGF-I; see Ref. 47), or systemic injection of N-methyl-d-aspartate (28). Thus the findings of this study are significantly important to support the concept that an increase in LHRH release is accompanied with an alteration of the timing of puberty.
The posterior hypothalamic lesion-induced increase in LHRH release was followed by precocious menarche. Although in our previous study posterior hypothalamic lesions advanced the timing of both menarche and first ovulation (42), in the present study the lesion did not result in early first ovulation. This difference does not appear to be because of the difference in the size and location of lesions within the hypothalamus, since they were comparable, but it may rather be attributed to the trend in our colony during the last two decades that menarche and first ovulation occur at much younger ages (Terasawa, unpublished observation). The cause of changes in the normal puberty age in our female rhesus colony remains to be investigated. Nonetheless, the absence of the advanced timing of first ovulation after precocious menarche with hypothalamic lesions is quite different from the precocious puberty induced by the infusion of the GABAA receptor antagonist bicuculline in the basal hypothalamus (15). Although in that study first ovulation occurred at the menarcheal age of control animals, in the present study first ovulation did not occur until the same age in sham/control animals, despite the fact that mean LHRH levels, pulse frequency, and pulse amplitude in lesion animals all increased after lesions.
The control mechanisms involved in the timing of first ovulation and menarche appear to differ, even though both require an increase in pulsatile LHRH release. Previously, we proposed a hypothesis that a larger amount of LHRH output from the hypothalamus is required for the positive-feedback effects of estrogen in the pubertal rhesus monkey (15). Consistent with the hypothesis, in the present study, we observed that mean LHRH levels dramatically increased after 35 mo of age in both lesion and sham/control animals (Figs. 6 and 8). Previous studies also suggest that, after an increase in pulsatile LHRH release (pulse frequency, pulse amplitude, and basal release) occurs at the onset of puberty, total output of LHRH release (pulse amplitude and basal release together) further increases between the early and midpubertal stages in female rhesus monkeys (7, 44). Moreover, escape from estrogen suppression or a decrease in hypersensitivity to estrogen in ovariectomized female monkeys occurred at the age of first ovulation, but not at the age of menarche (32, 46). Therefore, although lesions in the posterior hypothalamus can stimulate pulsatile LHRH release resulting precocious menarche, this procedure does not modify the mechanism responsible for the tempo of events leading to the entire process of puberty in primates.
Although in the previous study with ovariectomized monkeys posterior hypothalamic lesions advanced the age at which the positive feedback effect of estrogen on LH release occurs (15), in the present study in ovarian intact monkeys posterior hypothalamic lesions did not modify the age of first ovulation. This difference in ovariectomized and ovarian intact monkeys can be interpreted to mean that a small amount of circulating estrogen from the ovary after menarche in ovarian intact monkeys causes the restraint in the positive feedback mechanism on LH and/or LHRH release. The suppression of LH by a small elevation of estrogen from the ovary in leptin-treated female monkeys has been shown in a study by Wilson and colleagues (48). Again, these observations support the concept that the control mechanisms involved in the timing of first ovulation in normal puberty in primates differs from the mechanism initiating puberty.
A nocturnal elevation of GABA levels was seen in the lesion group at 15–17 mo of age. Previously, because we only examined GABA levels during the morning hours (0800 to 1100; see Ref. 21), we did not notice a daily GABA rhythm. A circadian variation in GABA release in the preoptic area of adult female rats (22) and a daily fluctuation of GABA content in the suprachiasmatic nucleus or hypothalamus in adult male rats (1, 5, 6) have been reported. Nonetheless, we need to conduct an additional study to establish a nocturnal elevation of GABA levels at 15–17 mo of age, since changes were not significant in sham/control animals.
The results of the present study also indicate that evening GABA levels in both lesion and sham/control groups at 18–20 mo were significantly lower than those at 15–17 mo. These results were quite surprising to us based on our previous observations (21, 41), since we expected that a high level of GABA would continue until very close to the early pubertal age. Although there is a possibility that sham lesion surgery with insertion of a lesion electrode in the posterior hypothalamus may have caused similar effects as lesions, further comparison of the results from two studies suggest that this difference may be the result of developmental changes between 15 and 17 mo and 18 and 20 mo of age. In the previous studies (21), GABA levels of prepubertal monkeys were measured at an average of 15.8 ± 1.1 mo of age (21), which is comparable to the prelesion age in the present study. Thus, although the developmental decrease in morning GABA levels does not occur until later age, it is possible that the developmental decrease in nocturnal GABA release occurs several months before the early pubertal stage, at 18–20 mo of age. If this assumption is correct, a difference in nocturnal GABA levels between the lesion and sham/control groups may have been seen if the lesion effects were examined shortly after lesions rather than a couple of months after lesions. The question of whether posterior hypothalamic lesions decrease GABA release in the S-ME leading to an increase in LHRH release remains to be resolved.
In contrast to the changes in evening GABA release, the lesion-induced LHRH increase appears to be associated with an increase in evening levels of glutamate. Evening glutamate levels at 18–20 mo of age in the lesion group were higher than those at 15–17 mo, whereas evening glutamate levels in the sham/lesion group at 18–20 mo of age did not differ from those at 15–17 mo. Corresponding to these glutamate changes, LHRH levels at 18–20 mo in the lesion group were higher than those at 15–17 mo of age, whereas this was not seen in the sham/control group. This observation underscores the significance of a pubertal increase in glutamate release and supports the hypothesis that glutamate in the hypothalamus plays a role in the mechanism of puberty (4, 28, 43).
A series of studies by Ojeda and colleagues (24, 25) have shown that growth factors, such as transforming growth factor-α (TGF-α), IGF-I, and fibroblast growth factor-1 from astroglia, are important for the mechanism of the onset of puberty. Hypothalamic expression of TGF-α receptor, erbB1, genes increases before puberty not only in rats but also in monkeys (16, 18, 19). Whereas transplantation of genetically engineered cells containing TGF-α causes precocious puberty (17, 31), the interference of TGF-α synthesis results in delayed puberty (19). Importantly, in female rats, lesions in the anterior hypothalamus resulting in precocious puberty induces extensive astrogliosis from which TGF-α is released (13). Astroglia also contain erbB1 receptors through which TGF-α in astroglia stimulate PGE2, resulting in LHRH release (14). Moreover, TGF-α mRNA expression in astroglia is observed in hypothalamic hamartomas removed from human patients exhibiting precocious puberty (12). Thus, in the present study, it is likely that posterior hypothalamic lesions induced astrogliosis, which might have released TGF-α, leading to LHRH release and triggering the initial phase of puberty, including changes in sex-skin swelling (presumably because of a small increase in circulating estrogen) and menarche. However, posterior hypothalamic lesions are not sufficient to result in a cascade of events necessary to advance the phase of puberty, since lesions did not alter the age of first ovulation. These results further suggest that modifying the timing of puberty by the placement of lesions in the hypothalamus is not a good tool for studying the mechanism of puberty in primates.
Stimulation of the glutamatergic neuronal system, and more recently stimulation of the kisspeptin/GPR54 neuronal system induce puberty in sexually immature rats and mice and pubertal changes in gonadotropin release in prepubertal male monkeys (4, 23, 28, 29, 36, 43). Similarly, electrical stimulation of the basal hypothalamus in female prepubertal monkeys, where LHRH neuroterminals are concentrated, results in an elevation of LHRH release (8). However, stimulation of the LHRH neuronal system directly or indirectly through excitation of upstream regulatory systems may not provide answers to the precise mechanism of the onset of puberty in primates. Unlike in rodents, the reduction of central GABAergic and/or neuropeptide Y inhibition is a prerequisite for the initiation and progression of spontaneous puberty in nonhuman primates (10, 26, 39). In the present study, the reduction in evening GABA levels had occurred at 18–20 mo of age, well before the early pubertal stage, although a causal relationship is not clearly established. Rather, a precocious increase in glutamate occurred in association with LHRH increase after hypothalamic lesions. Therefore, several critical questions remain to be answered. Does excitatory input, such as kisspeptin, give a signal for a reduction in the tonic GABA inhibition in the evening? Alternatively, does the developmental reduction in evening GABA inhibition precede an increase in other stimulatory neuromodulators, including kisspeptin? The investigation of the sequence of events in the basal hypothalamus will be very important for understanding the mechanism of the onset of puberty.
This work was supported by National Institutes of Health Grant R01-HD-11355. This publication was made possible in part by Grant P51 RR-000167 to the Wisconsin National Primate Research Center, University of Wisconsin-Madison.
We express sincere appreciation to Laure Lee Luchansky for HPLC analysis.
Present addresses: E. Kasuya, National Institute of Agrobiological Science, 2 Ikenodai, Tsukuba, Ibaraki, Japan (e-mail:); and M. Mizuno, Department of Brain Science and Engineering, Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology, 2–4 Hibikino, Wakamatsu, Kitakyushu, Japan (e-mail: ).
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- Copyright © 2007 by American Physiological Society