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In Vivo Gonadotropin-Releasing Hormone Secretion in Female Rats during Peripubertal Development and on Proestrus¹

Neuroscience Program and Department of Psychology, Michigan State University (C.L.S., H.N.R.), East Lansing, Michigan 48824; and Department of Neurobiology and Physiology, Northwestern University (P.E.C., J.E.L.), Evanston, Illinois 60208

Address all correspondence and requests for reprints to: Dr. Cheryl L. Sisk, Neuroscience Program, Michigan State University, East Lansing, Michigan 48824. E-mail: sisk{at}msu.edu

	Abstract

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Pubertal development in female rats is characterized by increased LH levels and the appearance of estrogen-dependent afternoon LH mini-surges. In these studies we performed the first analysis of GnRH patterns in peripubertal rats to determine whether there are similar changes in pulsatile GnRH release. Microdialysis samples were collected at 5-min intervals throughout a 5-h afternoon period from 22 rats sampled on a single day between 30–47 days of age. Adult female rats were sampled on proestrus for comparison. In 30- to 33-day-old rats, GnRH release was infrequent (2.7 pulses/5 h; n = 3), whereas intermediate pulse frequencies were observed in 34- to 37-day-old rats (6.4 pulses/5 h; n = 9) and 38- to 42-day-old (5.0 pulses/5 h; n = 5) rats. The highest GnRH pulse frequencies were observed in 43- to 47-day-old rats (9.4 pulses/5 h; n = 5). Mean GnRH pulse amplitude did not vary significantly with age. Animals sampled before vaginal opening (VO) exhibited significantly slower GnRH pulse frequencies than those sampled after vaginal opening (1.3 pulses/5 h pre-VO vs. 7.6 pulses/5 h post-VO; P = 0.01). An afternoon increase in GnRH secretion, defined operationally as a greater than 25% increase in mean GnRH levels in the last half of the sampling period and tentatively termed a mini-surge, was observed in 0%, 33%, 40%, and 60% of 30- to 33-, 34- to 37-, 38- to 42-, and 43- to 47-day-old rats, respectively. An overall increase in GnRH pulse frequency was observed in females displaying a mini-surge (9.0 pulses/5 h with mini-surge compared with 4.7 pulses/5 h with no mini-surge). The mini-surge itself, however, was associated with a late afternoon increase in GnRH pulse amplitude and not in pulse frequency. In adult proestrous rats, peak levels during the GnRH surge were an order of magnitude greater than those reached in pubertal animals. Our findings demonstrate that pubertal maturation in the female rat is associated with an acceleration of GnRH pulse generator activity and that later stages of pubertal maturation are characterized by the appearance of afternoon increases in GnRH release that may underlie previously reported mini-surges in LH.

	Introduction

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PUBERTAL DEVELOPMENT appears to be directed by progressive increases in the neurosecretory activity of GnRH neurons. In female rats (1), sheep (2), monkeys (3), and humans (4, 5), detailed analyses of peripubertal LH secretory patterns have been conducted to provide surrogate measures of GnRH release throughout pubertal maturation. These studies have revealed that the initial stages of pubertal maturation are mediated by an acceleration of GnRH pulse generator activity (GnRH pulse frequency), an increase in the amplitude of GnRH pulses, or both of these alterations in GnRH neurosecretion. Terasawa and colleagues have directly measured GnRH release patterns in female rhesus macaques and determined that all features of GnRH pulsatility (pulse amplitude, pulse frequency, and mean GnRH release) are significantly increased throughout pubertal maturation in this species (6).

It is not known whether the pubertal activation of pulsatile GnRH release occurs in rodents in a manner similar to that observed in monkeys. Previous studies have demonstrated that some features of this maturational process may indeed be different in rats compared with monkeys (1). For example, pulsatile LH secretions in pubertal rats (7) are increased in amplitude and mean level, but not in frequency, through the early stages of pubertal maturation. It is not clear whether this lack of an acceleration of LH pulsatility in pubertal rats represents a species-specific difference in GnRH neurosecretion at puberty or if the indirect measures of GnRH release used in the foregoing studies are unable to register an acceleration of the GnRH pulse generator that is not faithfully transduced by the anterior pituitary gland. The peripubertal female rat also appears to differ from its monkey counterpart in that it has been shown to secrete estrogen-dependent afternoon mini-surges of LH (7, 8). This phenomenon may represent an early signal for increased ovarian estrogen secretion, which would, in turn, contribute to the full development of the positive feedback stimulus directing release of gonadotropin surges in the adult. It has yet to be demonstrated that antecedent mini-surges of GnRH occur during this stage of sexual development.

In these studies we used an adaptation of the microdialysis approach (9) to analyze pulsatile GnRH release profiles in conscious, freely moving female rats at various stages of pubertal development. Our major aims were to determine 1) which features of GnRH release are altered throughout pubertal maturation of the female rat, 2) whether GnRH release profiles of the peripubertal female rat are characterized by afternoon increases in GnRH release, and 3) how afternoon patterns of GnRH secretion in peripubertal females compare with preovulatory GnRH surges in adult female rats.

	Materials and Methods

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Exp 1: peripubertal patterns of GnRH release
In vivo microdialysis was used to monitor the pattern of GnRH release in female rats at different ages spanning early to late pubertal development. Several cohorts of animals were studied separately at Northwestern University and Michigan State University (Table 1). To the extent possible, conditions at the two institutions were the same, including the light-dark cycle, housing, guide cannulas, microdialysis probes, and reagents. Minor differences in experimental protocols are noted below.

Experimental protocol. All experimental protocols were approved by institutional animal use and care committees. At ages ranging from 27–39 days, rats were anesthetized (ketamine hydrochloride, 80 mg/kg, im; xylazine, 10 mg/kg, im) and implanted with a guide cannula (CMA Microdialysis, Acton, MA) aimed 2 mm dorsal to the anterior aspect of the median eminence. The stereotaxic coordinates were 2.7 mm posterior to bregma, 0.3 mm lateral to the midsagittal suture, and 7.8 mm ventral to dura. The guide cannula was secured to the skull with jeweler’s screws and dental acrylic, and the scalp was sutured around the cannula assembly. A stylet was positioned in the guide cannula until the day of microdialysis, which was performed at least 2 days after stereotaxic surgery. Microdialysis probes (CMA Microdialysis) were 2 mm long and constructed with a polycarbonate membrane (20,000 Da cut-off). CMA 11 probes (0.24 mm diameter) were most often used, but during the course of this experiment CMA 11 probes were discontinued by the manufacturer, and thus CMA 12 probes (0.5 mm diameter) were used in 6 of 22 rats (Table 1). The use of CMA 12 probes was not confined to a single age group, nor was it associated with any trends toward increased or decreased GnRH pulse parameters or baseline levels compared with CMA 11 probes. On the day of sampling, the microdialysis probe was inserted into the guide cannula, and artificial cerebrospinal fluid (aCSF) was pumped through the probe at a rate of 1.6 µl/min for 1–2 h before sample collection began. Rats remained in their home cage during sampling. From 1300–1800 h, dialysate samples were collected as 5-min fractions (8-µl volume) into assay tubes on ice. Samples were immediately diluted with 92 µl assay buffer (0.1 M gel-PBS), vortexed, and snap-frozen in a dry ice-ethanol bath. Samples were stored at -80 C until RIA for GnRH was performed. At the end of the sampling period, the probe was removed, and the stylet was replaced. Rats were then lightly anesthetized with methoxyflurane (Metofane, Schering-Plough Corp., Union, NJ) and decapitated. Brains were removed, frozen on dry ice, and stored at -80 C until histology was performed to determine probe placement. Uteri were removed and weighed. In some cases (Michigan State), rats were anesthetized with sodium Nembutal (50 mg/kg, ip) at the end of the sampling period, the uteri were removed and weighed, and the animals were perfused intracardially with buffered saline and 4% formalin. The brains were postfixed overnight in 20% sucrose and 4% formalin. Brains were cut into 40-µm coronal sections and stored at -20 C in a polyethylene glycol-based cryoprotectant until histology was performed.

Exp 2: pattern of GnRH release on proestrus in adult females
In vivo microdialysis was used to monitor GnRH release on the day of proestrus in adult cycling females. This experiment was conducted at Northwestern University.

Animals and housing. Female rats, weighing 125 g, were obtained from Charles River Laboratories, Inc. They were housed three to five per cage before stereotaxic surgery and were singly housed afterward. Lights were on in the colony room for 14 h/day between 0500–1900 h Eastern Standard Time. Food and water were available ad libitum.

Experimental protocol. Ovarian cycles were monitored by daily assessment of vaginal cytology. Females showing at least three consecutive cycles were used for study. A guide cannula was stereotaxically implanted as described for Exp 1, except that stereotaxic coordinates were 3.0 mm posterior to bregma, 0.3 mm lateral to the midline, and 8 mm ventral to dura. Vaginal cytology was monitored daily after surgery to ensure that ovarian cycles continued. After at least three cycles postsurgery, microdialysis was performed on the day of proestrus in four individuals under the same conditions as those described for Exp 1. CMA 12 microdialysis probes were used in this experiment.

RIAs
GnRH was assayed in single dialysate samples using antirabbit GnRH EL-14 (provided by Dr. Oline Ronnekleiv, Oregon Health Sciences University, Portland, OR), iodinated GnRH, and ethanol precipitation of antibody-bound hormone. This assay has been previously validated (10). The limit of detection of the assay at 90% bound was either 0.11 pg/tube (Northwestern University) or 0.22 pg/tube (Michigan State). Intraassay coefficients of variation (CVs) ranged from 8–23% at Northwestern and from 16–25% at Michigan State. The interassay CVs were 27% and 28% at Northwestern and Michigan State, respectively. The intraassay CV for the appropriate assay was used for PULSAR analysis of individual hormone profiles (see below).

Histological assessment of probe placement
Fresh-frozen brains were cut on a cryostat into 30-µm coronal sections that were thaw-mounted onto gelatin-coated slides. Sections were fixed in 4% formalin, dehydrated in increasing concentrations of ethanol, and stained with thionin. Perfused brain sections were rinsed several times in 0.1 M phosphate buffer before being mounted onto slides and stained with thionin. The site of probe placement was determined by brightfield microscopic examination of tissue sections.

In Exp 1, microdialysis samples were obtained from 35 rats (22 at Northwestern and 13 at Michigan State). Microscopic analysis of brain sections from these rats revealed that the microdialysis probe was in or immediately lateral or anterior to the median eminence in 22 of these rats (14 at Northwestern and 8 at Michigan State). Figure 1 shows probe placement just lateral to the median eminence in a representative rat. In the remaining 13 rats, the probe was misplaced, and GnRH was undetectable in virtually all samples collected from these animals. Only data from the 22 rats with appropriately placed probes were used for statistical analysis.

View larger version (177K): [in this window] [in a new window]   Figure 1. Nissl-stained coronal section through the mediobasal hypothalamus showing microdialysis probe placement (arrow) just lateral to median eminence in a representative peripubertal female rat. 3V, Third ventricle; ME, median eminence; AP, anterior pituitary gland.

Analysis on the basis of age. All 22 rats in the study were used for this analysis. Rats were categorized into four age groups: 30–33 days (n = 3), 34–37 days (n = 9), 38–42 days (n = 5), and 43–47 days (n = 5). The number of GnRH pulses during the 5-h sampling period, mean pulse amplitude, mean GnRH levels (after normalizing assay sensitivity to 0.22 pg for all assays), and uterine weight were compared across the four age groups by one-way ANOVA.

Analysis on the basis of vaginal opening. Information on vaginal opening was available for 17 of the 22 rats in this study. Vaginal opening had occurred on or before the day of microdialysis sampling in 14 of these 17 rats. A t test was performed to determine whether the mean number of GnRH pulses or mean pulse amplitude was different before and after vaginal opening.

Analysis on the basis of occurrence of a late afternoon increase in GnRH. All 22 rats in the study were used for this analysis. Qualitative inspection of the GnRH profiles revealed a sustained late afternoon increase in GnRH in several individuals that was reminiscent of previously described peripubertal LH mini-surges (7). Rats were divided into 2 groups on the basis of whether there was a sustained increase in GnRH levels greater than 25% during the last half (1530–1800 h) of the sampling period compared with those in the first half (1300–1530 h). Eight rats met this operational criterion, and of those, 3 showed 50–100% increases in mean GnRH, and 3 others showed 100–500% increases in mean GnRH. We tentatively term these late afternoon increases in GnRH secretion GnRH mini-surges. The number of GnRH pulses per 5 h and mean pulse amplitude in females with and without a mini-surge were compared using unpaired t tests. To determine whether mini-surges were the result of late afternoon increases in GnRH pulse frequency, amplitude, or both, the number of pulses and mean pulse amplitude during the 2 halves of the sampling period were compared by paired t tests.

For all statistical analyses, differences were considered significant at P 0.05.

Exp 2. GnRH secretory profiles in proestrous females were analyzed by PULSAR, using the same G1–G5 values as in Exp 1. The mean number of pulses and mean pulse amplitude were determined from the PULSAR analysis.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experiment 1: peripubertal patterns of GnRH release
Analysis on the basis of age. Individual hormone profiles revealed a pulsatile pattern of GnRH release in the median eminence. Figure 2 shows GnRH secretory profiles of representative rats in the four age groups. An effect of age on the number of GnRH pulses during the 5-h sampling period approached, but did not achieve, statistical significance (F_3,18 = 2.89; P = 0.06; Fig. 3A). A direct comparison (unpaired t test) of the youngest and oldest age groups showed that the number of GnRH pulses during the 5-h sampling period was significantly greater in the 43- to 47-day-old rats compared with the 30- to 33-day-old rats (P = 0.03). Neither pulse amplitude (Fig. 3B) nor mean GnRH level (30–33 days, 0.224 ± 0.002 pg; 34–37 days, 0.481 ± 0.141 pg; 38–42 days, 0.313 ± 0.040 pg; 43–47 days, 0.401 ± 0.046 pg) was significantly affected by age. Mean uterine weight did not differ significantly among the four peripubertal age groups (30–33 days, 187.5 ± 31.5 mg; 34–37 days, 206.5 ± 50.9 mg; 38–42 days, 250.7± 40.9 mg; 43–47 days, 249.3 ± 52.2 mg).

View larger version (20K): [in this window] [in a new window]   Figure 2. GnRH secretory profiles of representative individual female rats in four age groups spanning pubertal development. Brackets over data points indicate GnRH pulses identified by PULSAR analysis.

View larger version (25K): [in this window] [in a new window]   Figure 3. Mean (±SEM) number of GnRH pulses (A) and mean (±SEM) GnRH pulse amplitude (B) during the 5-h sampling period in four age groups spanning pubertal development. Sample sizes for the age groups are n = 3 (30–33 days), n = 9 (34–37 days), n = 5 (38–42 days), and n = 5 (43–47 days). An asterisk indicates a significant difference in number of GnRH pulses per 5 h between the 43- to 47-day-old and 30- to 33-day-old groups (by unpaired t test, P = 0.03).

View larger version (21K): [in this window] [in a new window]   Figure 4. Mean (±SEM) number of GnRH pulses per 5 h in female rats sampled either before (n = 3) or after (n = 14) vaginal opening. An asterisk indicates a significant difference between the two groups.

View larger version (35K): [in this window] [in a new window]   Figure 5. GnRH secretory profiles of representative individual peripubertal female rats (N45, M6) displaying an afternoon GnRH mini-surge and individual adult female rats (A2 and A3) sampled during the afternoon of proestrus. Brackets over data points indicate GnRH pulses identified by PULSAR analysis. Note that the y-axis scale is an order of magnitude larger for adult proestrous females than that for peripubertal females displaying a mini-surge.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Methodological considerations
In vivo microdialysis allowed the first characterization of pubertal increases in GnRH pulsatile secretion in a female rodent model. Although microdialysis is clearly a powerful approach for the direct measurement of developmental changes in GnRH release, results should be viewed in the context of several methodological considerations.

Sensitivity. One limitation of microdialysis is the low recovery of peptide across the dialysis membrane, which for GnRH was determined to be 8–10% (Sisk, C. L., unpublished observations). This, combined with the low absolute amount of GnRH release expected in prepubertal animals, means that in many samples, GnRH levels were near the limits of detection of the RIA. Thus, we cannot rule out the possibility of GnRH pulse generator activity and low amplitude GnRH pulses in young animals that are below the sensitivity of microdialysis and the RIA.

Probe size. In Exp 1 it was necessary to use two differently sized microdialysis probes. Two lines of evidence suggest that probe diameter was not a significant factor in variation in parameters of GnRH secretory profiles. First, among the peripubertal females there was no apparent relationship between probe size and mean GnRH levels, pulse frequency, or amplitude. For example, both probe sizes were used within the group of females exhibiting GnRH mini-surges, which were associated with higher GnRH pulse frequency during the entire sampling period and late afternoon increases in pulse amplitude. Second, when features of GnRH secretion are compared in adult proestrous females (all sampled with the larger probe) and peripubertal females sampled with the larger probe, peak GnRH values were always an order of magnitude higher in the adults. Thus, a larger probe size was not invariably associated with higher measurements of GnRH.

Probe site. Some variation in probe placement is unavoidable. Probe placement in these experiments was restricted to the median eminence and its lateral or anterior aspects, through which GnRH axons converge on their way to the median eminence. A previous examination of the influence of push-pull perfusion cannula placement on GnRH secretory patterns in the monkey found that GnRH pulse frequency was consistent across cannula tip sampling sites, but that pulse amplitude varied in a manner that reflected the distribution of GnRH fibers (12). Thus, it is possible that variability in probe placement contributed to some variability in GnRH pulse amplitude in the present study. However, higher pulse amplitude was not always associated with probe placement closer to the median eminence, and a range of mean pulse amplitudes was observed across animals with comparable probe placement. It is important to note that the late afternoon increase in pulse amplitude that was characteristic of GnRH mini-surges occurred within the sampling period, and therefore is clearly not attributable to variation in probe site.

Physiological considerations
Chronological age and GnRH secretion. Pulse frequency significantly increased with chronological age when only the youngest and oldest age groups were directly compared. The lack of a significant effect of age when ANOVA was performed with all age groups is related in part to the small sample size in the youngest age group, intermediate pulse frequencies in the two middle age groups, and within age group variability. In addition to the methodological considerations previously discussed, within age group variability is also probably a function of two biological factors. First, there is inherent individual variation in the age at onset of puberty. Second, as ovarian cyclicity emerges in later stages of puberty, and estrous cyclicity was not monitored after vaginal opening in this experiment, older females of the same chronological age may have varied in endocrine status on the day of sampling. This is supported by the observation that the occurrence of late afternoon increases in GnRH was not uniformly observed in all individuals in the older age groups. These biological sources of variability probably contributed to the lack of a significant effect of age on uterine weight as well. Thus, chronological age is an imperfect predictor of pubertal and reproductive status.

Vaginal opening and GnRH secretion. GnRH pulse frequency was unambiguously increased after vaginal opening, whereas pulse amplitude was virtually identical before and after vaginal opening. As vaginal opening is estrogen dependent, these results link increased GnRH pulse frequency with ovarian maturation.

Comparison of peripubertal patterns of GnRH and LH release
A detailed analysis of pulsatile LH secretion (7) previously demonstrated that LH pulse amplitude is greater in 30- to 37-day-old rats compared with those at 27–29 days of age. In the present study we were not able to monitor GnRH release in animals as young as 27–29 days of age, so a comparison of pulsatile GnRH and LH determinations in the two studies cannot be made. It remains possible, therefore, that an early increase in GnRH pulse amplitude may also occur as a component of the pubertal initiation process before 30 days of age. It is also possible that the ability of the pituitary gland to transduce a GnRH stimulus is progressively enhanced as the GnRH pulse generator activity accelerates; thus, the observed increase in LH pulse amplitude demonstrated in the foregoing study may not have been reflective of an increase in the amplitude of underlying GnRH release. Indeed, pulsatile GnRH stimulation has been used to intentionally increase the responsiveness of the pituitary in peripubertal monkeys and thereby allow a more sensitive bioassay of endogenous GnRH pulsatility (13). Endogenous GnRH pulsatility may likewise arouse the pituitary gland from a relatively refractory state during the earliest stages of puberty, and this process would present itself as a progressive increase in LH pulse amplitude, even if the antecedent GnRH stimulation were of a relatively consistent amplitude. Two mechanisms by which a pubertal increase in GnRH pulse frequency could lead to increased LH pulse amplitude include enhanced expression of GnRH receptors in gonadotropes (14) and preferential synthesis of LH over that of FSH, leading to increased pituitary stores of LH (15).

In a subset of the animals examined, late afternoon increases in GnRH secretion were noted in which there were 3- to 4-fold increases in the amplitude of GnRH pulses for a period of 2–3 h. This pattern of GnRH closely resembles that of LH mini-surges previously observed in comparably aged rats. LH mini-surges are characterized by afternoon increases in LH pulse amplitude, but not frequency (8). Moreover, GnRH pulse amplitude during the late afternoon increases in secretion represent approximately 10% of the peak levels during proestrous surges in the adult, a proportion remarkably consistent with that observed for the comparison of LH mini-surges and adult LH surges (1). Therefore, we have tentatively termed these late afternoon increases in GnRH secretion mini-surges, with two caveats. First, we could not monitor GnRH and LH simultaneously in these peripubertal animals, and we do not know whether GnRH mini-surges, like LH mini-surges, are estrogen-dependent events. Second, the pattern of late afternoon GnRH secretion was not always characterized by a distinct ascending and descending component (compare rat N45 with M6, Fig. 5). The emergence of daily afternoon increases in LH secretion is characteristic of early pubertal development in female rats and hamsters (7, 16). Thus, the patterns of afternoon GnRH secretion observed in this study may reflect a gradual progression from daily afternoon increases in pulse amplitude to estrogen-dependent mini-surges that eventually give rise to estrogen- dependent LH surges. Simultaneous measurements of GnRH and LH and experimental manipulation of circulating estrogen will be required to confirm whether this is the case.

Implications for pubertal activation of the hypothalamic-pituitary-gonadal axis
Both GnRH messenger RNA and protein are expressed within GnRH neurons of the female rat before the onset of puberty, indicating the potential for secretion of the peptide even in neonatal animals (17, 18). Increases in GnRH messenger RNA and puberty onset can both be accelerated in juvenile female rats by activation of N-methyl-D-aspartic acid receptors (18, 19, 20). Together with these studies, our demonstration of an increase in GnRH pulse frequency related to age, vaginal opening, and the occurrence of an afternoon mini-surge suggest that the rate-limiting feature for the pubertal increase in gonadotropin secretion may be the electrophysiological activation of GnRH cells. In the prepubertal animal, GnRH cells may be relatively refractory to excitation, or they may not receive appropriate excitatory signals. Alternatively, inhibitory inputs to GnRH neurons may reduce the responsiveness of the cells to excitatory signals. The onset of puberty may thus be prompted by an increase in GnRH neuronal excitability, an increase in the magnitude of excitatory afferent signals, and/or a decrease in the magnitude of inhibitory afferent signals.

It is likely that extrahypothalamic signals, such as metabolic and sensory cues, also figure importantly in the pubertal activation of the GnRH pulse generator. Food restriction suppresses LH pulsatility and prevents maturation in rats (21), indicating that the pubertal activation of the GnRH pulse generator cannot fully occur under conditions of prolonged negative energy balance. Return to ad libitum feeding quickly leads to a restoration of LH pulsatility and the development of the sexually mature state (21). It is not clear how metabolic factors may permit maturation of the GnRH pulse generator, but these signals may include leptin (22, 23), insulin (24), GH (25), insulin-like growth factor (26), glucose (27, 28), or some combination of these and other indicators of energetic state or fuel availability (29). The acceleration of GnRH pulsatility during puberty may also be gated by neuroendocrine signals mediating photoperiodic cues (30, 31) and other sensory stimuli (32, 33).

Conclusions
Taken together, our two major findings, increased GnRH pulsatility and the occurrence of afternoon increases in GnRH pulse amplitude, generally support the model elaborated by Ojeda and colleagues for the neuroendocrine processes mediating pubertal maturation in female rats (1). They have proposed that the end of the juvenile period of development is characterized by an increase in pulsatile GnRH secretion, which prompts an increase in LH secretion and ovarian estrogen production. The increase in estrogen secretion, in turn, is believed to evoke the release of afternoon mini-surges of GnRH and LH. Our experiments have provided the first direct confirmation of the two essential elements of this theory, namely, that GnRH pulsatility is increased, and that late afternoon increases in GnRH pulse amplitude occur with greater frequency during this transitional stage. The cellular and molecular mechanisms mediating the pubertal acceleration of GnRH pulsatility and the stimulation of putative GnRH mini-surges remain to be resolved.

	Acknowledgments

 
We thank Jane Venier and Jeffrey Norgle for technical assistance.

	Footnotes

 
¹ This work was supported by NIH Grant HD-26483 and NSF Grant IBN 96–02169 (to C.L.S.) and by NIH Grants R01-HD-20677 and P01-HD-21921 (to J.E.L.).

Received November 17, 2000.

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