Pubertal Acceleration of Pulsatile Gonadotropin-Releasing Hormone Release in Male Rats as Revealed by Microdialysis
Abstracte@qg, 百拇医药
A microdialysis technique was used in male rats to directly assess the postulate that pubertal maturation is associated with accelerated GnRH pulsatility. Juvenile male rats, postnatal d 43 or 45 (n = 4) were stereotaxically fitted with guide cannulas directed toward the lateral median eminence, and repeated microdialysis experiments were conducted over 4–6 d. In each session, samples were collected continuously over 12 h (0900–2100 h) at 5-min intervals Results from individual peripubertal animals were pooled into two time bins for postnatal d 45–47 and 48–50, respectively, and GnRH characteristics were compared between the two epochs. The GnRH pulse frequency and mean GnRH concentration were significantly elevated at 48–50 d compared with 45–47 d. The GnRH pulsatility characteristics for 45–47 d vs. 48–50 d were as follows: pulse frequency, 0.74 ± 0.16 vs. 1.79 ± 0.19 pulses/h (P < 0.05); pulse amplitude, 254.1 ± 22.3 vs. 347.2 ± 15.8 pg/ml (difference in value from trough to peak); and mean release, 0.55 ± 0.03 vs. 2.04 ± 0.04 pg/5 min (P < 0.05). An additional two rats were dialyzed only once on postnatal d 50 to assess the effects of repeated sampling; the GnRH pulse characteristics in these animals were similar to those in rats sampled for a third or fourth time on postnatal d 48–50. To further assess the possible effects of repeated sampling on GnRH release profiles, a group of adult male rats (postnatal d 95–105; n = 3) was also dialyzed on four consecutive days. In these rats no significant alteration in GnRH pulse generator activity was observed over the four sessions. Moreover, the increase in GnRH pulse frequency observed in the peripubertal rats was found to be sustained in adult animals. To better understand the temporal relationship of GnRH pulse generator activity to reproductive maturation, groups of male rats were killed from postnatal d 45–56 along with an adult group at 95–105 d (n = 5/group) and examined for physiological signs of reproductive development. Gradual increases in serum levels of LH and testosterone and decreases in FSH and inhibin B were seen from postnatal d 45–56 to adulthood. Mature spermatozoa were found in the vas deferens by postnatal d 53. Our results demonstrate that in the late juvenile stage of male rat development, GnRH pulse generator activity is gradually accelerated over the course of consecutive days. This acceleration occurs over a period during which serum LH and testosterone are rising to adult levels, and it precedes the presence of mature spermatozoa in the vas deferens by 3 d. Our observations provide direct support for the hypothesis that an acceleration of GnRH pulsatility is the critical neural stimulus for the initiation of pubertal maturation in males. The peripheral and central cues that prompt the pubertal activation of the GnRH pulse generator remain to be characterized.
Introductionn8;, 百拇医药
PUBERTY IS a period of development in which a complex series of physiological events leads to the establishment of reproductive capacity. An early and key component of this process appears to be an increase in LH secretion, which, in turn, is probably driven by an acceleration of pulsatile GnRH release into the hypophysial portal vasculature (1, 2, 3, 4, 5). This activation of the GnRH pulse generator remains poorly understood, in large part due to the technical difficulties inherent in the longitudinal analysis of GnRH release patterns. Studies providing direct analyses of peripubertal GnRH pulsatility in female mammals have been few (5, 6), and no such studies have been performed in the male of any species. In this report we provide the first direct characterization of peripubertal GnRH release in a male animal and assess the alterations in GnRH pulsatility that may be most critically important for maturation of the male reproductive axis.n8;, 百拇医药
Pubertal alterations in GnRH pulsatility have been directly assessed in female rhesus monkeys (5) and rats (6). Watanabe and Terasawa (5) used a push-pull perfusion technique to show an increase in pulsatile GnRH release, pulse frequency, and pulse amplitude during the early pubertal stage before menarche in the female rhesus monkey. In the female rat, Sisk et al. (6) used the microdialysis technique to demonstrate that GnRH pulse generator activity, in particular the aspect of pulse frequency, is accelerated with increasing age and after vaginal opening.l, 百拇医药
Other studies have implicated a role for increased GnRH release during male sexual development. In primates, a pronounced rise in gonadotropin levels occurs during sexual maturation (1, 7). This increase in gonadotropin secretion takes place in the absence of the testes (8), providing further evidence for a change in the central nervous system-pituitary mechanism regulating gonadotropin secretion at the time of puberty. In a related study Suter et al. (9) used the GnRH-primed, in situ pituitary of the agonadal male rhesus monkey as an in vivo biological assay of GnRH activity before and during puberty. They demonstrated relatively rapid nocturnal shifts from low to high frequency LH release in the majority of pubertal animals, implicating a correlative change in GnRH secretion. To date, however, there have been no studies that have directly examined GnRH release in males during the peripubertal period.
In the present study we sought to determine 1) the changing dynamics of GnRH pulse frequency, pulse amplitude, and/or mean release rate in individual male rats throughout the course of puberty; 2) whether the GnRH release profile is changed abruptly or gradually; and 3) the time course of any alterations in GnRH activity in relation to other physiological changes associated with puberty. To address these questions, we used a microdialysis approach for high resolution monitoring of GnRH release. This method allows for remote sampling for prolonged periods and repeated sessions over consecutive days under conditions of normal activity and minimal stress. Our measurements provide the first comprehensive characterization of GnRH pulse generator activity during puberty in a male animal. The results of this study clearly implicate GnRH pulse frequency regulation as a primary neural signal for the pubertal maturation of the male reproductive axis.7, 百拇医药
Materials and Methods
Animals and surgical protocolezw, 百拇医药
All experimental procedures were conducted with the approval of Northwestern University’s animal care and use committee. Male Sprague Dawley rats (Charles River Laboratories, Inc., Portage, WI) were housed before surgeries in groups of four or five rats per cage in a temperature-controlled room, with lights on from 0500–1900 h. Animals had access to tap water and laboratory chow ad libitum. After surgeries, rats were housed individually, but otherwise under the same conditions.ezw, 百拇医药
Experiment 1ezw, 百拇医药
Four juvenile male rats were selected for repeated 12-h microdialysis sessions. Two of these animals were sampled on postnatal d 47 (P47), P48, P49, and P50; a third was sampled on P45, P46, P48, and P49; and a fourth was sampled on P45, P46, P47, and P50. Two additional rats were sampled during one session only, on P50, to assess whether GnRH levels in microdialysates may be subject to artifactual alteration with repeated sampling. For the same reason, three adult male rats, ranging in age from P95–P105, were also sampled on four consecutive days.
Two days before start of microdialysis experiments, rats were anesthetized with ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip) and mounted in a stereotaxic frame. A microdialysis probe guide cannula (Carnegie-Medicin, Acton, MA) was implanted into each rat, with the tip targeted to a position just above the median eminence. The cannula was cemented in place with skull screws and dental acrylic. The coordinates used were anterior/posterior, -2.9; medial/lateral, 0.3; and dorsal/ventral, -8.1, relative to bregma (10).[?7as;., http://www.100md.com
On the day of an experiment, a microdialysis probe (14-mm stainless steel shaft, polycarbonate-polyether membrane of 2-mm length and 0.5-mm diameter, 3-µl internal volume; CMA/12, CMA, Acton, MA) was inserted into the guide at 0800 h, and the flow of artificial cerebrospinal fluid (6) was allowed to equilibrate for 1 h at a rate of 2.0 µl/min. Thereafter, from 0900–2100 h, 10-µl samples were collected every 5 min into glass test tubes in a fraction collector as previously described (6). PBS with 0.1% gelatin was added to each tube to bring the volume up to 100 µl, and the tubes were snap-frozen in ethanol and dry-ice. All samples were stored at -80 C until RIA for GnRH.
At the end of an experiment, rats were anesthetized, and a 1% toluidine blue solution was injected into the guide cannula through a modified probe. After decapitation, the brain was rapidly removed, snap-frozen on dry-ice, and sliced on a cryotome. Fifteen-micrometer sections were mounted on slides, stained with cresyl violet, and examined under a microscope to document the placement of the microdialysis probe.)2, 百拇医药
Experiment 2)2, 百拇医药
To determine the time course of developmental changes in the reproductive axis throughout puberty, groups of five rats per day from P45–P56 along with a group of adult males (P95–P100) were anesthetized via methoxyfluorane inhalation and immediately killed by decapitation from 0900–1000 h. Trunk blood was collected and centrifuged, and the plasma was stored at -20 C for subsequent RIA of LH, FSH, testosterone (T), and inhibin B. Sperm counts were determined as follows. Each rat’s right vas deferens was removed, and the contents were squeezed into a sterile 35-mm tissue culture dish (Corning, Inc., Corning, NY) containing 2 ml M2 conditioned medium (Sigma, St. Louis, MO) at 37 C and gently mixed. Thereafter, both chambers of a hemocytometer were filled with the solution, and spermatozoa were counted using a phase contrast microscope (Nikon, Melville, NY). Sperm counts were calculated as the average of two chambers x 104, x 2 (diluted in 2 ml medium). Both testes were removed, weighed, and immersed in Bouin’s fixative. After dehydration in alcohol, testes were embedded in paraffin (TissuePrep2, Fisher Scientific, Pittsburgh, PA), sectioned on a microtome, and stained with hematoxylin and eosin for histological analysis.
RIAs3:, 百拇医药
LH, FSH, and T in plasma and GnRH in microdialysates were determined by RIA. Inhibin B was measured using an ELISA assay kit (Serotec, Raleigh, NC). All samples from the same experiment were measured in a single assay. Reagents for RIAs of LH and FSH were obtained from the NIDDK. The LH assay used the LH RP-3 standard and had a lower limit of detection of 0.1 ng/ml and an intraassay coefficient of variation of 6%. For the FSH RIA, the FSH RP-2 standard preparation was used, and the intraassay coefficient of variation was 7.5%. RIA of T was performed using kits obtained from ICN Biochemicals, Inc. (Costa Mesa, CA), with an intraassay coefficient of variation of 11%. The RIA of GnRH in microdialysates was performed using the EL-14 antiserum provided by Dr. Martin Kelly (Oregon Health Sciences University, Portland, OR). The GnRH RIA had a lower limit of sensitivity of 0.1 pg/tube. The intraassay coefficient of variation was 13% for the range of concentrations that included the majority of the GnRH values in microdialysates. The average interassay coefficient of variation for the assay was 11%.
Statistics@&phq, http://www.100md.com
In Experiment 1 significant pulses of GnRH were identified, and pulse characteristics were determined with the ULTRA software program of Van Cauter (11). A threshold of two times the coefficient of variation in the corresponding ranges of values in the GnRH RIA was used in the algorithm program to identify significant GnRH pulses. For each of the four juvenile animals that were sampled on several days, the average GnRH pulse amplitude (difference in value from peak to trough), GnRH pulse frequency, and mean GnRH concentration were determined for each of two time bins; the first included all data from sessions conducted from P45–P47, and the second included all data obtained from P48–P50. For each of the three GnRH release characteristics, the values for P45–P47 were then compared with those for P48–P50 by means of paired t tests. Results were considered significant if P < 0.05. The GnRH release characteristics in adult male rats were similarly analyzed to determine whether GnRH release parameters were altered on d 3–4 vs. d 1–2 during four consecutive days of dialysis sessions. For hormone data obtained in experiment 2, the mean ± SE hormone levels were determined for each age group, and significant differences among age groups were assessed by one-way ANOVA, followed by Tukey’s post hoc test. Results were again considered significant at P < 0.05.
Resultsx@, 百拇医药
Experiment 1x@, 百拇医药
Figure 1, A and B, and Fig. 2, A and B, show 12-h GnRH release profiles obtained on four sequential days (ages P45–P50) in four different individual peripubertal male rats. Microdialysis samples were obtained from a probe directed lateral to the median eminence, at 5-min intervals from 0900–2100 h. GnRH release was pulsatile on all days studied as has been shown for LH secretion in juvenile males (12).x@, 百拇医药
fig.ommitteedx@, 百拇医药
Figure 1. Longitudinal pulsatile GnRH release measurements in two individual peripubertal male rats. A, Measurements made in one male rat on P45, P46, P47, and P50. B, Data from a second peripubertal male rat on P45, P46, P48, and P49, as indicated. In each experimental session, microdialysis samples were taken every 5 min for 12 h, from 0900–2100 h, on the postnatal days indicated. Samples were analyzed by GnRH RIA. In this and subsequent figures, significant pulses as identified by ULTRA pulse analysis are indicated ().
fig.ommitteedi8\uw{t, 百拇医药
Figure 2. Pulsatile GnRH release profiles from two additional peripubertal male rats (A and B) on P47–P50.i8\uw{t, 百拇医药
For all animals a gradual shift to an accelerated GnRH secretion pattern was evident over consecutive days of measurement. Analysis of pulses in the animal whose GnRH profiles are depicted in Fig. 1A revealed a gradual increase from a mean pulse frequency of 0.333 pulses/h and a mean pulse amplitude of 20.86 ± 14.32 pg/ml at P45 (top panel) to 2.083 pulses/h and 123.82 ± 12.664 pg/ml at P50 (bottom panel). A similar gradual increase in GnRH pulse generator activity was seen in the profiles depicted in Fig. 1B. In this animal GnRH pulsatility progressed from a mean pulse frequency of 1.083 pulses/h and a mean pulse amplitude of 117.13 ± 16.66 pg/ml on P45 (top panel) to 2.083 pulses/h and 113.37 ± 15.28 pg/ml on P48. On P49 the GnRH pulse frequency in this animal (1.42 pulses/h) remained higher than that observed on P45, although it was somewhat reduced from that on the previous day.
In the animal illustrated in Fig. 3A, the GnRH profile on the first day of sampling (P47) consisted of only one significant pulse, of relatively small amplitude (<25 pg/ml/12 h). Thereafter, pulse generator activity gradually increased on each day, culminating on P50 with a pulse frequency of 2.58 pulses/h and a mean pulse amplitude of 142.46 ± 15.84 pg/ml. Depicted in Fig. 2B are GnRH profiles from the only animal in which pulsatile GnRH release was found to accelerate and then exhibit a pronounced deceleration on the last day of sampling. Here, GnRH secretory activity progressed from a mean pulse frequency of 0.716 pulses/h with a mean pulse amplitude of 60.14 pg/ml on P47 (Fig. 2B, top panel) to 2.25 pulses/h and 99.93 ± 11.14 pg/ml on P48 (Fig. 2B, second panel), eaking on P49 with 2.33 pulses/h and 161.11 ± 13.526 pg/ml (Fig. 2B, third panel), only to fall again on P50 to 0.33 pulses/h and 61.07 ± 9.75 pg/ml (Fig. 2B, fourth panel).q, 百拇医药
fig.ommitteedq, 百拇医药
Figure 3. Summary statistics for GnRH release profiles in four peripubertal rats. The GnRH pulse frequency, GnRH pulse amplitude, and mean GnRH concentration were calculated for each animal during the P45–P47 and P48–P50 age bins, and the summary statistics for GnRH release characteristics in each of the two periods are depicted as the mean ± SE. Paired t tests revealed a significant increase in GnRH pulse frequency and mean release in the P48–P50 vs. the P45–47 period. *, Significant difference at P < 0.05.*8, http://www.100md.com
The data from Figs. 1 and 2 were combined to produce the summary GnRH pulse characteristics depicted in Fig. 3 Values from the four individual animals were combined into bins of ages P45–P47 and P48–P50. The GnRH pulsatility characteristics for 45–47 vs. 48–50 d were as follows: pulse frequency, 0.74 ± 0.16 vs. 1.79 ± 0.19 pulses/h (P < 0.05); pulse amplitude, 254.1 ± 22.3 vs. 347.2 ± 15.8 pg/ml; and mean release, 0.55 ± 0.03 vs. 2.04 ± 0.04 pg/5 min (P < 0.05). Thus, significant increases in mean pulse frequency and mean GnRH concentration were seen in animals from the P45–P47 to the P48–P50 period. A rise in GnRH pulse amplitude approached, but did not meet, the criterion for statistical significance.
As patterns of GnRH release may have been artifactually influenced by the dialysis procedure itself over the course of multiple dialysis sessions, we compared GnRH profiles observed in animals dialyzed for the first time on P50 with those in peripubertal animals dialyzed during a fourth consecutive day on P49 or P50. The GnRH release profiles of the two peripubertal animals that were dialyzed once only, on P50, are depicted in Fig. 4. Both animals exhibited GnRH pulse frequencies (2.5 and 2.7 pulses/h) and mean GnRH pulse amplitudes (115.5 and 273.3 pg/ml) similar to those in animals dialyzed for the third or fourth time on P49 or P50.#mr, 百拇医药
fig.ommitteed#mr, 百拇医药
Figure 4. Pulsatile GnRH release profiles from two additional male rats (A and B) on P50. In both rats, microdialysis samples were taken exactly as in previous experiments, except that the rats were sampled on a single day rather than on consecutive days.#mr, 百拇医药
A group of three intact adult male rats (P95–P105) were dialyzed for 6 h on four consecutive days. Representative GnRH pulse profiles from one animal are shown in Fig. 5A and the summary statistics for the pulsatile GnRH release characteristics in all three adult animals are shown in Fig. 5B. No significant changes in mean GnRH pulse frequency, mean GnRH pulse amplitude, or mean GnRH release were observed in these animals throughout the four sequential days of dialysis sampling. The mean GnRH pulse frequencies observed on all days in the adult animals closely matched those attained by P48–P50 in the peripubertal animals (Fig. 3). Mean GnRH pulse amplitude and mean GnRH concentration, by contrast, were substantially lower in the adult animals (Fig. 5B) vs. the peripubertal animals (Fig. 3).
fig.ommitteedx.\h&, http://www.100md.com
Figure 5. Longitudinal analysis of GnRH release patterns in adult male rats. A, Pulsatile GnRH release profiles from an individual adult male rat. Microdialysis samples were obtained at 5-min intervals for 6 h in four separate microdialysis sessions conducted on four consecutive days. B, Summary statistics for GnRH release profiles determined in microdialysis sessions conducted on four consecutive days in three adult male rats. Data are presented as the mean ± SE GnRH pulse frequency, GnRH pulse amplitude, and mean GnRH concentration on each day. No significant changes in GnRH pulse characteristics were observed over the course of 4 d of sampling.x.\h&, http://www.100md.com
Experiment 2x.\h&, http://www.100md.com
From P45 through P56 five rats per day were killed, and several different aspects of physiology were analyzed and compared over the peripubertal period. A group of five adult male rats was also included in this analysis. Gradual increases with age in mean body weight, mean epididymal weight, and mean testis weight are shown in Table 1, corresponding to the pubertal growth spurt in accessory organ weight documented in previous reports (13, 14). Histological analysis of tubular sections from fixed testicular tissue showed no detectable change in the volume of seminiferous epithelium from rats aged P45 or more. Similarly, some mature spermatids were present within the lumen of tubules from all animals studied.
fig.ommitteedau+c}8!, 百拇医药
Table 1. Mean ± SE body weights, epididymus weights, and testes weights in cohorts of male rats that were anesthetized and killed at 0900–1000 h on the days indicatedau+c}8!, 百拇医药
Mean LH, FSH, T, and inhibin B levels from male rats aged P45–P54 and an adult group aged P95–P100 are shown in Fig. 6. Levels of LH rose gradually from P45–P54, with levels in the P95–P100 animals being significantly higher than those observed in P45 rats (P < 0.001), whereas adult levels of FSH were significantly lower than those in all but P52–P54 rats. Rats at P52, P53, and P55 and adult rats had significantly higher levels of T than those at P45. Adult rats had significantly lower levels of inhibin B than peripubertal animals (P < 0.001).au+c}8!, 百拇医药
fig.ommitteedau+c}8!, 百拇医药
Figure 6. Hormone levels in blood obtained from peripubertal and adult male rats in experiment 2. Data are presented as the mean ± SE (n = 5/group). a, P < 0.05 vs. P95–P100; a+, P < 0.001 vs. P95–P100; b, P < 0.05 vs. P45; b+, P < 0.001 vs. P45.
Figure 7 shows the results of sperm count analyses from rat vas deferens throughout the peripubertal period. Small numbers of round spermatids, classified as step 1–6 spermatids (15), were observed in the vas deferens fluid of rats as early as P48. Mature, post-step 15 sperm were not seen in the majority of vas deferens until P51 (Fig. 7A). Significant numbers of mature sperm were not seen until P53 (Fig. 7B).*, http://www.100md.com
fig.ommitteed*, http://www.100md.com
Figure 7. Analysis of sperm in the vas deferens of cohorts of rats killed on P45–P56 or as adults. A, Results are plotted as the percentage of rats exhibiting mature sperm present in the vas deferens at each day of age. B, Results are plotted as the mean ± SE number of mature sperm in vas deferens at each day of age.*, http://www.100md.com
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Discussion*, http://www.100md.com
It is generally held that the initiation of puberty in male mammals is mediated by an activation of the neuronal substrates governing pulsatile GnRH neurosecretion. In male rodents (3) and monkeys (9) and in pubertal boys (16), analyses of pulsatile LH secretion have strongly supported the idea that accelerated GnRH pulse generator activity evokes peripubertal increases in gonadotropin secretion, which, in turn, prompt gonadal steroidogenesis and sexual maturation. In the present study we tested this hypothesis directly using a microdialysis approach (17) to obtain the first measurements of GnRH pulse generator activity in peripubertal male rats. Our results unequivocally demonstrate that GnRH pulsatility accelerates throughout pubertal development, plateauing to a rate of pulse generation that appears to be maintained in the adult male rat. These observations provide direct support for the contention that an increase in GnRH pulse generator activity, specifically an increase in GnRH pulse frequency, is a key neural signal for maturation of the reproductive hormonal axis in male rats.
In the present study the frequency of pulsatile GnRH release, as measured in hypothalamic dialysates, was found to approximately double in peripubertal male rats from the ages of P45–P50. The accelerated rate observed in P48–P50 rats was similar to the GnRH pulse frequency in adult rats. The amplitude of GnRH pulses also tended to be greater in the later pubertal time period; however, this trend did not reach statistical significance. Measurements of serum LH and T in companion cohorts of animals revealed that mean levels of both of these hormones also increase developmentally from P45 to adulthood. Our measurements of LH and T are consistent with the results of previous studies that similarly documented the occurrence of increases in serum T and LH secretion (18, 19, 20, 21) throughout the peripubertal period. It is generally believed that the pubertal rise in T is dependent upon a rise in LH levels, and that the pubertal rise in LH is dependent upon increased GnRH secretion. These assumptions are strongly supported by the observations that intermittent GnRH stimulation or treatment with the GnRH secretagogue N-methyl-D,L-aspartic acid (NMA), can prematurely initiate the pubertal process in rodents (22) and monkeys (23, 24). Moreover, spontaneous increases in LH secretion, presumably driven by increased GnRH release, have been documented in castrate peripubertal animals in which circulating T levels were clamped at unvarying levels (3). Taken together, the weight of evidence clearly supports the view that an acceleration of GnRH pulsatility is the proximal signal for the pubertal rise in LH and, hence, T secretion.
Our longitudinal analyses of GnRH pulsatility in individual male rats revealed that the pubertal activation of GnRH pulsatility is principally manifest as continuous increases in GnRH pulse frequency and mean release. In some cases, however, GnRH pulse frequency and mean release appeared to wane for several hours before resuming an upward trajectory in release rate. Indeed, in one animal GnRH pulsatility steadily increased over the course of three consecutive days and then returned to a substantially lower level throughout d 4 of sampling. The latter phenomenon was similarly observed in one subject in a previously published analysis of pulsatile LH secretion in pubertal male monkeys (9). This waxing and occasional waning of GnRH pulsatility and the relatively shallow rise in LH levels during the peripubertal period are consistent with the idea that puberty in the male rat involves the establishment of new set points for both feedforward and feedback controls within the reproductive axis. Thus, the acquisition of adult patterns of GnRH, LH, and T secretion may proceed through several iterations of the following sequence of events: 1) an acceleration of the GnRH pulse generator occurs over the course of several days during the late juvenile period; 2) increased GnRH pulsatility prompts a corresponding increase in LH secretion, which, in turn, evokes an increase in gonadal T production; 3) increased T secretion provides an enhanced negative feedback signal to the pituitary gland (25, 26) and to the GnRH pulse generator (27); 4) GnRH and LH secretion temporarily wanes as a result of increased T feedback; and 5) GnRH pulsatility and LH secretion resume their upward trajectory. Successive periods of waxing and waning ultimately culminate in the attainment of an adult level of GnRH pulsatility, LH secretion, and T output in the context of a reequilibrated homeostatic control system.
How does the foregoing model account for the developmental patterns of FSH and inhibin B secretions? In the male rat serum concentrations of FSH appear to reach a peak by P30–P40 and thereafter fall gradually to a stable, and relatively low, adult level (18, 20, 28). Developmental changes in serum inhibin B levels essentially mirror those of FSH (29), and are probably a reflection of the degree of FSH stimulatory support (30). Thus, in the male rat serum FSH levels peak and then begin to decline before serum LH levels rise, indicating the existence of a mechanism permitting differential regulation of the gonadotropins. It has been demonstrated in a variety of experimental and physiological circumstances that alterations in the frequency of pulsatile GnRH stimulation can differentially affect the rate of LH and FSH secretion (31, 32, 33, 34). Thus, one explanation for the developmental divergence of FSH and LH secretions is that increasingly higher rates of endogenous GnRH pulse generation may increasingly favor release of LH vs. FSH from pituitary gonadotropes as sexual maturation proceeds. The underlying cellular mechanisms that may mediate differential sensitivities of FSH and LH to varying frequencies of GnRH stimulation remain to be clarified.
Apart from the pubertal rise in GnRH pulse frequency, an increase in baseline GnRH levels was also observed in the later pubertal sampling periods (P48–P50). The mean GnRH level in microdialysates obtained during this time was higher than those seen at earlier times and in the adult male rats. These peripubertal increases in mean GnRH levels could not be attributed to increased permeability of microdialysis membranes with repeated use, as no probe was used in more than two sessions, and exchange tests performed in vitro have not revealed any variation in probe performance that could account for these results. We did, however, consider it possible that the accumulated effects of repeated GnRH microdialysis sessions in an individual animal may have artifactually induced this increase in mean GnRH level, perhaps through repeated disturbance of the extracellular compartment about the probe tip.j!s9|:, http://www.100md.com
To ascertain whether this was the case, we performed repeated microdialysis sessions in adult male rats to determine whether mean GnRH levels were successively increased in these animals throughout the same schedule of experiments. The results clearly demonstrate that repeated microdialysis samplings per se do not induce increases in mean GnRH levels in microdialysates. As an additional control, we also compared GnRH release characteristics in rats sampled only once, on d 50 of postnatal life, with the data obtained from animals sampled for the fourth time on the same day; no differences in GnRH mean levels were observed. We therefore consider it unlikely that the peripubertal rise in mean GnRH levels occurred in these experiments as an artifactual product of the microdialysis procedures.
The rise and then apparent fall in mean GnRH levels throughout puberty may instead reflect a physiological accumulation of GnRH in the extracellular compartment that occurs during the pubertal acceleration of GnRH pulsatility. Although the reasons for this are not immediately clear, it is possible that these peripubertal changes in mean GnRH levels reflect alterations in GnRH synthesis rates during this developmental period. The content of GnRH peptide in hypothalamic extracts is increased throughout postnatal life, reaching a peak level at 45 d and then declining thereafter in male rats (35). In female rats an increase, followed by a decrease, in GnRH tissue content occur earlier (35) and appears to be associated with an increase in cytoplasmic GnRH mRNA, but not GnRH primary transcript (36). It is thus possible that an increase in the rate of posttranscriptional processing during the peripubertal period may lead to increased GnRH production and increased mass of peptide produced and released. The decline in GnRH levels in the adult, however, is not easily explained in this scenario.
Alternatively, the rise in mean GnRH levels in peripubertal animals may be explained by alterations in GnRH proteolytic degradation. The GnRH decapeptide is processed to peptide subproducts, e.g. GnRH (1, 2, 3, 4, 5), primarily through the actions of prolyl endopeptidase and endopeptidase 24.15, and recent work has demonstrated that prolyl endopeptidase activity in particular is decreased during the late juvenile period (37). Thus, an acceleration of GnRH pulsatility during the juvenile-pubertal transition may be unaccompanied by concomitant increases in endogenous peptidase activity, thereby leading to an accumulation of GnRH in the extracellular compartment. Subsequently, the return of mean GnRH levels to lower values in the adult rat could be explained by a possible increase in extracellular degradation due to a return to higher levels of peptidase activity.l}p-g-], 百拇医药
Our observations support the contention that an acceleration of GnRH pulse generator activity functions as the major neural signal for the initiation of puberty in male rats (3), and thus our findings are in direct agreement with those obtained via pulsatile LH measurements in male monkeys (9) and boys (16). It is clear, nevertheless, that there may be major differences in the control of GnRH pulsatility in juvenile rodents compared with primates. The castration of juvenile male monkeys, for example, does not produce a significant increase in GnRH pulse generator activity, whereas in the male rat the same treatment does evoke increased pulsatile LH (and presumably GnRH) secretion. It is thus believed that the GnRH pulse generator in the juvenile monkey is completely or nearly dormant, and hence refractory to the negative feedback actions of gonadal hormones, whereas in the male rat GnRH pulsatility occurs, albeit at low levels, during the late juvenile period. Does this indicate a fundamental difference in the events underlying puberty in the two species? Repeated injection of the GnRH secretagogue, NMA, can induce pubertal maturation prematurely in both monkeys (24) and rats (22), suggesting that changes in GnRH pulse generator activity that drive puberty may differ quantitatively, but perhaps not qualitatively. It is possible that the abbreviated life span of the rat compared with the monkey necessarily compresses the same (or similar) events into a shorter developmental window of time. A dormant period for the GnRH pulse generator may therefore precede the period over which our measurements were made, and the acceleration of GnRH pulse generator activity may commence earlier as well. It is also possible that the GnRH pulse generator is not completely inactive in male primates, as was thought to be the case from early studies in monkeys (9). Indeed, the use of sensitive LH assays in boys has revealed the presence of diminished, yet detectable, pulsatile LH baselines before puberty (16).
The present observations taken together with the previous finding that NMA injections induce precocious puberty (22) suggest that the rate-limiting feature for the pubertal activation of gonadotropin secretion is the electrophysiological activation of GnRH neurons. The molecular and cellular mechanisms that mediate the pubertal activation of the pulse-generating mechanism are not completely understood, but probably depend upon the combined effects of 1) metabolic cues, such as leptin, insulin, GH, IGF, and glucose (38); 2) sensory cues, such as photoperiodic stimuli (39); 3) neuroglial-derived growth factors (40); and/or 4) intrahypothalamic actions of inhibitory and stimulatory neurotransmitters. The mechanisms by which these signals are transduced and integrated to yield an acceleration of GnRH pulsatility remain to be determined.k1elgt/, http://www.100md.com
Acknowledgmentsk1elgt/, http://www.100md.com
We gratefully acknowledge the technical expertise of Ms. Brigitte Mann.k1elgt/, http://www.100md.com
Received July 31, 2002.
Accepted for publication October 3, 2002.s., 百拇医药
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A microdialysis technique was used in male rats to directly assess the postulate that pubertal maturation is associated with accelerated GnRH pulsatility. Juvenile male rats, postnatal d 43 or 45 (n = 4) were stereotaxically fitted with guide cannulas directed toward the lateral median eminence, and repeated microdialysis experiments were conducted over 4–6 d. In each session, samples were collected continuously over 12 h (0900–2100 h) at 5-min intervals Results from individual peripubertal animals were pooled into two time bins for postnatal d 45–47 and 48–50, respectively, and GnRH characteristics were compared between the two epochs. The GnRH pulse frequency and mean GnRH concentration were significantly elevated at 48–50 d compared with 45–47 d. The GnRH pulsatility characteristics for 45–47 d vs. 48–50 d were as follows: pulse frequency, 0.74 ± 0.16 vs. 1.79 ± 0.19 pulses/h (P < 0.05); pulse amplitude, 254.1 ± 22.3 vs. 347.2 ± 15.8 pg/ml (difference in value from trough to peak); and mean release, 0.55 ± 0.03 vs. 2.04 ± 0.04 pg/5 min (P < 0.05). An additional two rats were dialyzed only once on postnatal d 50 to assess the effects of repeated sampling; the GnRH pulse characteristics in these animals were similar to those in rats sampled for a third or fourth time on postnatal d 48–50. To further assess the possible effects of repeated sampling on GnRH release profiles, a group of adult male rats (postnatal d 95–105; n = 3) was also dialyzed on four consecutive days. In these rats no significant alteration in GnRH pulse generator activity was observed over the four sessions. Moreover, the increase in GnRH pulse frequency observed in the peripubertal rats was found to be sustained in adult animals. To better understand the temporal relationship of GnRH pulse generator activity to reproductive maturation, groups of male rats were killed from postnatal d 45–56 along with an adult group at 95–105 d (n = 5/group) and examined for physiological signs of reproductive development. Gradual increases in serum levels of LH and testosterone and decreases in FSH and inhibin B were seen from postnatal d 45–56 to adulthood. Mature spermatozoa were found in the vas deferens by postnatal d 53. Our results demonstrate that in the late juvenile stage of male rat development, GnRH pulse generator activity is gradually accelerated over the course of consecutive days. This acceleration occurs over a period during which serum LH and testosterone are rising to adult levels, and it precedes the presence of mature spermatozoa in the vas deferens by 3 d. Our observations provide direct support for the hypothesis that an acceleration of GnRH pulsatility is the critical neural stimulus for the initiation of pubertal maturation in males. The peripheral and central cues that prompt the pubertal activation of the GnRH pulse generator remain to be characterized.
Introductionn8;, 百拇医药
PUBERTY IS a period of development in which a complex series of physiological events leads to the establishment of reproductive capacity. An early and key component of this process appears to be an increase in LH secretion, which, in turn, is probably driven by an acceleration of pulsatile GnRH release into the hypophysial portal vasculature (1, 2, 3, 4, 5). This activation of the GnRH pulse generator remains poorly understood, in large part due to the technical difficulties inherent in the longitudinal analysis of GnRH release patterns. Studies providing direct analyses of peripubertal GnRH pulsatility in female mammals have been few (5, 6), and no such studies have been performed in the male of any species. In this report we provide the first direct characterization of peripubertal GnRH release in a male animal and assess the alterations in GnRH pulsatility that may be most critically important for maturation of the male reproductive axis.n8;, 百拇医药
Pubertal alterations in GnRH pulsatility have been directly assessed in female rhesus monkeys (5) and rats (6). Watanabe and Terasawa (5) used a push-pull perfusion technique to show an increase in pulsatile GnRH release, pulse frequency, and pulse amplitude during the early pubertal stage before menarche in the female rhesus monkey. In the female rat, Sisk et al. (6) used the microdialysis technique to demonstrate that GnRH pulse generator activity, in particular the aspect of pulse frequency, is accelerated with increasing age and after vaginal opening.l, 百拇医药
Other studies have implicated a role for increased GnRH release during male sexual development. In primates, a pronounced rise in gonadotropin levels occurs during sexual maturation (1, 7). This increase in gonadotropin secretion takes place in the absence of the testes (8), providing further evidence for a change in the central nervous system-pituitary mechanism regulating gonadotropin secretion at the time of puberty. In a related study Suter et al. (9) used the GnRH-primed, in situ pituitary of the agonadal male rhesus monkey as an in vivo biological assay of GnRH activity before and during puberty. They demonstrated relatively rapid nocturnal shifts from low to high frequency LH release in the majority of pubertal animals, implicating a correlative change in GnRH secretion. To date, however, there have been no studies that have directly examined GnRH release in males during the peripubertal period.
In the present study we sought to determine 1) the changing dynamics of GnRH pulse frequency, pulse amplitude, and/or mean release rate in individual male rats throughout the course of puberty; 2) whether the GnRH release profile is changed abruptly or gradually; and 3) the time course of any alterations in GnRH activity in relation to other physiological changes associated with puberty. To address these questions, we used a microdialysis approach for high resolution monitoring of GnRH release. This method allows for remote sampling for prolonged periods and repeated sessions over consecutive days under conditions of normal activity and minimal stress. Our measurements provide the first comprehensive characterization of GnRH pulse generator activity during puberty in a male animal. The results of this study clearly implicate GnRH pulse frequency regulation as a primary neural signal for the pubertal maturation of the male reproductive axis.7, 百拇医药
Materials and Methods
Animals and surgical protocolezw, 百拇医药
All experimental procedures were conducted with the approval of Northwestern University’s animal care and use committee. Male Sprague Dawley rats (Charles River Laboratories, Inc., Portage, WI) were housed before surgeries in groups of four or five rats per cage in a temperature-controlled room, with lights on from 0500–1900 h. Animals had access to tap water and laboratory chow ad libitum. After surgeries, rats were housed individually, but otherwise under the same conditions.ezw, 百拇医药
Experiment 1ezw, 百拇医药
Four juvenile male rats were selected for repeated 12-h microdialysis sessions. Two of these animals were sampled on postnatal d 47 (P47), P48, P49, and P50; a third was sampled on P45, P46, P48, and P49; and a fourth was sampled on P45, P46, P47, and P50. Two additional rats were sampled during one session only, on P50, to assess whether GnRH levels in microdialysates may be subject to artifactual alteration with repeated sampling. For the same reason, three adult male rats, ranging in age from P95–P105, were also sampled on four consecutive days.
Two days before start of microdialysis experiments, rats were anesthetized with ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip) and mounted in a stereotaxic frame. A microdialysis probe guide cannula (Carnegie-Medicin, Acton, MA) was implanted into each rat, with the tip targeted to a position just above the median eminence. The cannula was cemented in place with skull screws and dental acrylic. The coordinates used were anterior/posterior, -2.9; medial/lateral, 0.3; and dorsal/ventral, -8.1, relative to bregma (10).[?7as;., http://www.100md.com
On the day of an experiment, a microdialysis probe (14-mm stainless steel shaft, polycarbonate-polyether membrane of 2-mm length and 0.5-mm diameter, 3-µl internal volume; CMA/12, CMA, Acton, MA) was inserted into the guide at 0800 h, and the flow of artificial cerebrospinal fluid (6) was allowed to equilibrate for 1 h at a rate of 2.0 µl/min. Thereafter, from 0900–2100 h, 10-µl samples were collected every 5 min into glass test tubes in a fraction collector as previously described (6). PBS with 0.1% gelatin was added to each tube to bring the volume up to 100 µl, and the tubes were snap-frozen in ethanol and dry-ice. All samples were stored at -80 C until RIA for GnRH.
At the end of an experiment, rats were anesthetized, and a 1% toluidine blue solution was injected into the guide cannula through a modified probe. After decapitation, the brain was rapidly removed, snap-frozen on dry-ice, and sliced on a cryotome. Fifteen-micrometer sections were mounted on slides, stained with cresyl violet, and examined under a microscope to document the placement of the microdialysis probe.)2, 百拇医药
Experiment 2)2, 百拇医药
To determine the time course of developmental changes in the reproductive axis throughout puberty, groups of five rats per day from P45–P56 along with a group of adult males (P95–P100) were anesthetized via methoxyfluorane inhalation and immediately killed by decapitation from 0900–1000 h. Trunk blood was collected and centrifuged, and the plasma was stored at -20 C for subsequent RIA of LH, FSH, testosterone (T), and inhibin B. Sperm counts were determined as follows. Each rat’s right vas deferens was removed, and the contents were squeezed into a sterile 35-mm tissue culture dish (Corning, Inc., Corning, NY) containing 2 ml M2 conditioned medium (Sigma, St. Louis, MO) at 37 C and gently mixed. Thereafter, both chambers of a hemocytometer were filled with the solution, and spermatozoa were counted using a phase contrast microscope (Nikon, Melville, NY). Sperm counts were calculated as the average of two chambers x 104, x 2 (diluted in 2 ml medium). Both testes were removed, weighed, and immersed in Bouin’s fixative. After dehydration in alcohol, testes were embedded in paraffin (TissuePrep2, Fisher Scientific, Pittsburgh, PA), sectioned on a microtome, and stained with hematoxylin and eosin for histological analysis.
RIAs3:, 百拇医药
LH, FSH, and T in plasma and GnRH in microdialysates were determined by RIA. Inhibin B was measured using an ELISA assay kit (Serotec, Raleigh, NC). All samples from the same experiment were measured in a single assay. Reagents for RIAs of LH and FSH were obtained from the NIDDK. The LH assay used the LH RP-3 standard and had a lower limit of detection of 0.1 ng/ml and an intraassay coefficient of variation of 6%. For the FSH RIA, the FSH RP-2 standard preparation was used, and the intraassay coefficient of variation was 7.5%. RIA of T was performed using kits obtained from ICN Biochemicals, Inc. (Costa Mesa, CA), with an intraassay coefficient of variation of 11%. The RIA of GnRH in microdialysates was performed using the EL-14 antiserum provided by Dr. Martin Kelly (Oregon Health Sciences University, Portland, OR). The GnRH RIA had a lower limit of sensitivity of 0.1 pg/tube. The intraassay coefficient of variation was 13% for the range of concentrations that included the majority of the GnRH values in microdialysates. The average interassay coefficient of variation for the assay was 11%.
Statistics@&phq, http://www.100md.com
In Experiment 1 significant pulses of GnRH were identified, and pulse characteristics were determined with the ULTRA software program of Van Cauter (11). A threshold of two times the coefficient of variation in the corresponding ranges of values in the GnRH RIA was used in the algorithm program to identify significant GnRH pulses. For each of the four juvenile animals that were sampled on several days, the average GnRH pulse amplitude (difference in value from peak to trough), GnRH pulse frequency, and mean GnRH concentration were determined for each of two time bins; the first included all data from sessions conducted from P45–P47, and the second included all data obtained from P48–P50. For each of the three GnRH release characteristics, the values for P45–P47 were then compared with those for P48–P50 by means of paired t tests. Results were considered significant if P < 0.05. The GnRH release characteristics in adult male rats were similarly analyzed to determine whether GnRH release parameters were altered on d 3–4 vs. d 1–2 during four consecutive days of dialysis sessions. For hormone data obtained in experiment 2, the mean ± SE hormone levels were determined for each age group, and significant differences among age groups were assessed by one-way ANOVA, followed by Tukey’s post hoc test. Results were again considered significant at P < 0.05.
Resultsx@, 百拇医药
Experiment 1x@, 百拇医药
Figure 1, A and B, and Fig. 2, A and B, show 12-h GnRH release profiles obtained on four sequential days (ages P45–P50) in four different individual peripubertal male rats. Microdialysis samples were obtained from a probe directed lateral to the median eminence, at 5-min intervals from 0900–2100 h. GnRH release was pulsatile on all days studied as has been shown for LH secretion in juvenile males (12).x@, 百拇医药
fig.ommitteedx@, 百拇医药
Figure 1. Longitudinal pulsatile GnRH release measurements in two individual peripubertal male rats. A, Measurements made in one male rat on P45, P46, P47, and P50. B, Data from a second peripubertal male rat on P45, P46, P48, and P49, as indicated. In each experimental session, microdialysis samples were taken every 5 min for 12 h, from 0900–2100 h, on the postnatal days indicated. Samples were analyzed by GnRH RIA. In this and subsequent figures, significant pulses as identified by ULTRA pulse analysis are indicated ().
fig.ommitteedi8\uw{t, 百拇医药
Figure 2. Pulsatile GnRH release profiles from two additional peripubertal male rats (A and B) on P47–P50.i8\uw{t, 百拇医药
For all animals a gradual shift to an accelerated GnRH secretion pattern was evident over consecutive days of measurement. Analysis of pulses in the animal whose GnRH profiles are depicted in Fig. 1A revealed a gradual increase from a mean pulse frequency of 0.333 pulses/h and a mean pulse amplitude of 20.86 ± 14.32 pg/ml at P45 (top panel) to 2.083 pulses/h and 123.82 ± 12.664 pg/ml at P50 (bottom panel). A similar gradual increase in GnRH pulse generator activity was seen in the profiles depicted in Fig. 1B. In this animal GnRH pulsatility progressed from a mean pulse frequency of 1.083 pulses/h and a mean pulse amplitude of 117.13 ± 16.66 pg/ml on P45 (top panel) to 2.083 pulses/h and 113.37 ± 15.28 pg/ml on P48. On P49 the GnRH pulse frequency in this animal (1.42 pulses/h) remained higher than that observed on P45, although it was somewhat reduced from that on the previous day.
In the animal illustrated in Fig. 3A, the GnRH profile on the first day of sampling (P47) consisted of only one significant pulse, of relatively small amplitude (<25 pg/ml/12 h). Thereafter, pulse generator activity gradually increased on each day, culminating on P50 with a pulse frequency of 2.58 pulses/h and a mean pulse amplitude of 142.46 ± 15.84 pg/ml. Depicted in Fig. 2B are GnRH profiles from the only animal in which pulsatile GnRH release was found to accelerate and then exhibit a pronounced deceleration on the last day of sampling. Here, GnRH secretory activity progressed from a mean pulse frequency of 0.716 pulses/h with a mean pulse amplitude of 60.14 pg/ml on P47 (Fig. 2B, top panel) to 2.25 pulses/h and 99.93 ± 11.14 pg/ml on P48 (Fig. 2B, second panel), eaking on P49 with 2.33 pulses/h and 161.11 ± 13.526 pg/ml (Fig. 2B, third panel), only to fall again on P50 to 0.33 pulses/h and 61.07 ± 9.75 pg/ml (Fig. 2B, fourth panel).q, 百拇医药
fig.ommitteedq, 百拇医药
Figure 3. Summary statistics for GnRH release profiles in four peripubertal rats. The GnRH pulse frequency, GnRH pulse amplitude, and mean GnRH concentration were calculated for each animal during the P45–P47 and P48–P50 age bins, and the summary statistics for GnRH release characteristics in each of the two periods are depicted as the mean ± SE. Paired t tests revealed a significant increase in GnRH pulse frequency and mean release in the P48–P50 vs. the P45–47 period. *, Significant difference at P < 0.05.*8, http://www.100md.com
The data from Figs. 1 and 2 were combined to produce the summary GnRH pulse characteristics depicted in Fig. 3 Values from the four individual animals were combined into bins of ages P45–P47 and P48–P50. The GnRH pulsatility characteristics for 45–47 vs. 48–50 d were as follows: pulse frequency, 0.74 ± 0.16 vs. 1.79 ± 0.19 pulses/h (P < 0.05); pulse amplitude, 254.1 ± 22.3 vs. 347.2 ± 15.8 pg/ml; and mean release, 0.55 ± 0.03 vs. 2.04 ± 0.04 pg/5 min (P < 0.05). Thus, significant increases in mean pulse frequency and mean GnRH concentration were seen in animals from the P45–P47 to the P48–P50 period. A rise in GnRH pulse amplitude approached, but did not meet, the criterion for statistical significance.
As patterns of GnRH release may have been artifactually influenced by the dialysis procedure itself over the course of multiple dialysis sessions, we compared GnRH profiles observed in animals dialyzed for the first time on P50 with those in peripubertal animals dialyzed during a fourth consecutive day on P49 or P50. The GnRH release profiles of the two peripubertal animals that were dialyzed once only, on P50, are depicted in Fig. 4. Both animals exhibited GnRH pulse frequencies (2.5 and 2.7 pulses/h) and mean GnRH pulse amplitudes (115.5 and 273.3 pg/ml) similar to those in animals dialyzed for the third or fourth time on P49 or P50.#mr, 百拇医药
fig.ommitteed#mr, 百拇医药
Figure 4. Pulsatile GnRH release profiles from two additional male rats (A and B) on P50. In both rats, microdialysis samples were taken exactly as in previous experiments, except that the rats were sampled on a single day rather than on consecutive days.#mr, 百拇医药
A group of three intact adult male rats (P95–P105) were dialyzed for 6 h on four consecutive days. Representative GnRH pulse profiles from one animal are shown in Fig. 5A and the summary statistics for the pulsatile GnRH release characteristics in all three adult animals are shown in Fig. 5B. No significant changes in mean GnRH pulse frequency, mean GnRH pulse amplitude, or mean GnRH release were observed in these animals throughout the four sequential days of dialysis sampling. The mean GnRH pulse frequencies observed on all days in the adult animals closely matched those attained by P48–P50 in the peripubertal animals (Fig. 3). Mean GnRH pulse amplitude and mean GnRH concentration, by contrast, were substantially lower in the adult animals (Fig. 5B) vs. the peripubertal animals (Fig. 3).
fig.ommitteedx.\h&, http://www.100md.com
Figure 5. Longitudinal analysis of GnRH release patterns in adult male rats. A, Pulsatile GnRH release profiles from an individual adult male rat. Microdialysis samples were obtained at 5-min intervals for 6 h in four separate microdialysis sessions conducted on four consecutive days. B, Summary statistics for GnRH release profiles determined in microdialysis sessions conducted on four consecutive days in three adult male rats. Data are presented as the mean ± SE GnRH pulse frequency, GnRH pulse amplitude, and mean GnRH concentration on each day. No significant changes in GnRH pulse characteristics were observed over the course of 4 d of sampling.x.\h&, http://www.100md.com
Experiment 2x.\h&, http://www.100md.com
From P45 through P56 five rats per day were killed, and several different aspects of physiology were analyzed and compared over the peripubertal period. A group of five adult male rats was also included in this analysis. Gradual increases with age in mean body weight, mean epididymal weight, and mean testis weight are shown in Table 1, corresponding to the pubertal growth spurt in accessory organ weight documented in previous reports (13, 14). Histological analysis of tubular sections from fixed testicular tissue showed no detectable change in the volume of seminiferous epithelium from rats aged P45 or more. Similarly, some mature spermatids were present within the lumen of tubules from all animals studied.
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Table 1. Mean ± SE body weights, epididymus weights, and testes weights in cohorts of male rats that were anesthetized and killed at 0900–1000 h on the days indicatedau+c}8!, 百拇医药
Mean LH, FSH, T, and inhibin B levels from male rats aged P45–P54 and an adult group aged P95–P100 are shown in Fig. 6. Levels of LH rose gradually from P45–P54, with levels in the P95–P100 animals being significantly higher than those observed in P45 rats (P < 0.001), whereas adult levels of FSH were significantly lower than those in all but P52–P54 rats. Rats at P52, P53, and P55 and adult rats had significantly higher levels of T than those at P45. Adult rats had significantly lower levels of inhibin B than peripubertal animals (P < 0.001).au+c}8!, 百拇医药
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Figure 6. Hormone levels in blood obtained from peripubertal and adult male rats in experiment 2. Data are presented as the mean ± SE (n = 5/group). a, P < 0.05 vs. P95–P100; a+, P < 0.001 vs. P95–P100; b, P < 0.05 vs. P45; b+, P < 0.001 vs. P45.
Figure 7 shows the results of sperm count analyses from rat vas deferens throughout the peripubertal period. Small numbers of round spermatids, classified as step 1–6 spermatids (15), were observed in the vas deferens fluid of rats as early as P48. Mature, post-step 15 sperm were not seen in the majority of vas deferens until P51 (Fig. 7A). Significant numbers of mature sperm were not seen until P53 (Fig. 7B).*, http://www.100md.com
fig.ommitteed*, http://www.100md.com
Figure 7. Analysis of sperm in the vas deferens of cohorts of rats killed on P45–P56 or as adults. A, Results are plotted as the percentage of rats exhibiting mature sperm present in the vas deferens at each day of age. B, Results are plotted as the mean ± SE number of mature sperm in vas deferens at each day of age.*, http://www.100md.com
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Discussion*, http://www.100md.com
It is generally held that the initiation of puberty in male mammals is mediated by an activation of the neuronal substrates governing pulsatile GnRH neurosecretion. In male rodents (3) and monkeys (9) and in pubertal boys (16), analyses of pulsatile LH secretion have strongly supported the idea that accelerated GnRH pulse generator activity evokes peripubertal increases in gonadotropin secretion, which, in turn, prompt gonadal steroidogenesis and sexual maturation. In the present study we tested this hypothesis directly using a microdialysis approach (17) to obtain the first measurements of GnRH pulse generator activity in peripubertal male rats. Our results unequivocally demonstrate that GnRH pulsatility accelerates throughout pubertal development, plateauing to a rate of pulse generation that appears to be maintained in the adult male rat. These observations provide direct support for the contention that an increase in GnRH pulse generator activity, specifically an increase in GnRH pulse frequency, is a key neural signal for maturation of the reproductive hormonal axis in male rats.
In the present study the frequency of pulsatile GnRH release, as measured in hypothalamic dialysates, was found to approximately double in peripubertal male rats from the ages of P45–P50. The accelerated rate observed in P48–P50 rats was similar to the GnRH pulse frequency in adult rats. The amplitude of GnRH pulses also tended to be greater in the later pubertal time period; however, this trend did not reach statistical significance. Measurements of serum LH and T in companion cohorts of animals revealed that mean levels of both of these hormones also increase developmentally from P45 to adulthood. Our measurements of LH and T are consistent with the results of previous studies that similarly documented the occurrence of increases in serum T and LH secretion (18, 19, 20, 21) throughout the peripubertal period. It is generally believed that the pubertal rise in T is dependent upon a rise in LH levels, and that the pubertal rise in LH is dependent upon increased GnRH secretion. These assumptions are strongly supported by the observations that intermittent GnRH stimulation or treatment with the GnRH secretagogue N-methyl-D,L-aspartic acid (NMA), can prematurely initiate the pubertal process in rodents (22) and monkeys (23, 24). Moreover, spontaneous increases in LH secretion, presumably driven by increased GnRH release, have been documented in castrate peripubertal animals in which circulating T levels were clamped at unvarying levels (3). Taken together, the weight of evidence clearly supports the view that an acceleration of GnRH pulsatility is the proximal signal for the pubertal rise in LH and, hence, T secretion.
Our longitudinal analyses of GnRH pulsatility in individual male rats revealed that the pubertal activation of GnRH pulsatility is principally manifest as continuous increases in GnRH pulse frequency and mean release. In some cases, however, GnRH pulse frequency and mean release appeared to wane for several hours before resuming an upward trajectory in release rate. Indeed, in one animal GnRH pulsatility steadily increased over the course of three consecutive days and then returned to a substantially lower level throughout d 4 of sampling. The latter phenomenon was similarly observed in one subject in a previously published analysis of pulsatile LH secretion in pubertal male monkeys (9). This waxing and occasional waning of GnRH pulsatility and the relatively shallow rise in LH levels during the peripubertal period are consistent with the idea that puberty in the male rat involves the establishment of new set points for both feedforward and feedback controls within the reproductive axis. Thus, the acquisition of adult patterns of GnRH, LH, and T secretion may proceed through several iterations of the following sequence of events: 1) an acceleration of the GnRH pulse generator occurs over the course of several days during the late juvenile period; 2) increased GnRH pulsatility prompts a corresponding increase in LH secretion, which, in turn, evokes an increase in gonadal T production; 3) increased T secretion provides an enhanced negative feedback signal to the pituitary gland (25, 26) and to the GnRH pulse generator (27); 4) GnRH and LH secretion temporarily wanes as a result of increased T feedback; and 5) GnRH pulsatility and LH secretion resume their upward trajectory. Successive periods of waxing and waning ultimately culminate in the attainment of an adult level of GnRH pulsatility, LH secretion, and T output in the context of a reequilibrated homeostatic control system.
How does the foregoing model account for the developmental patterns of FSH and inhibin B secretions? In the male rat serum concentrations of FSH appear to reach a peak by P30–P40 and thereafter fall gradually to a stable, and relatively low, adult level (18, 20, 28). Developmental changes in serum inhibin B levels essentially mirror those of FSH (29), and are probably a reflection of the degree of FSH stimulatory support (30). Thus, in the male rat serum FSH levels peak and then begin to decline before serum LH levels rise, indicating the existence of a mechanism permitting differential regulation of the gonadotropins. It has been demonstrated in a variety of experimental and physiological circumstances that alterations in the frequency of pulsatile GnRH stimulation can differentially affect the rate of LH and FSH secretion (31, 32, 33, 34). Thus, one explanation for the developmental divergence of FSH and LH secretions is that increasingly higher rates of endogenous GnRH pulse generation may increasingly favor release of LH vs. FSH from pituitary gonadotropes as sexual maturation proceeds. The underlying cellular mechanisms that may mediate differential sensitivities of FSH and LH to varying frequencies of GnRH stimulation remain to be clarified.
Apart from the pubertal rise in GnRH pulse frequency, an increase in baseline GnRH levels was also observed in the later pubertal sampling periods (P48–P50). The mean GnRH level in microdialysates obtained during this time was higher than those seen at earlier times and in the adult male rats. These peripubertal increases in mean GnRH levels could not be attributed to increased permeability of microdialysis membranes with repeated use, as no probe was used in more than two sessions, and exchange tests performed in vitro have not revealed any variation in probe performance that could account for these results. We did, however, consider it possible that the accumulated effects of repeated GnRH microdialysis sessions in an individual animal may have artifactually induced this increase in mean GnRH level, perhaps through repeated disturbance of the extracellular compartment about the probe tip.j!s9|:, http://www.100md.com
To ascertain whether this was the case, we performed repeated microdialysis sessions in adult male rats to determine whether mean GnRH levels were successively increased in these animals throughout the same schedule of experiments. The results clearly demonstrate that repeated microdialysis samplings per se do not induce increases in mean GnRH levels in microdialysates. As an additional control, we also compared GnRH release characteristics in rats sampled only once, on d 50 of postnatal life, with the data obtained from animals sampled for the fourth time on the same day; no differences in GnRH mean levels were observed. We therefore consider it unlikely that the peripubertal rise in mean GnRH levels occurred in these experiments as an artifactual product of the microdialysis procedures.
The rise and then apparent fall in mean GnRH levels throughout puberty may instead reflect a physiological accumulation of GnRH in the extracellular compartment that occurs during the pubertal acceleration of GnRH pulsatility. Although the reasons for this are not immediately clear, it is possible that these peripubertal changes in mean GnRH levels reflect alterations in GnRH synthesis rates during this developmental period. The content of GnRH peptide in hypothalamic extracts is increased throughout postnatal life, reaching a peak level at 45 d and then declining thereafter in male rats (35). In female rats an increase, followed by a decrease, in GnRH tissue content occur earlier (35) and appears to be associated with an increase in cytoplasmic GnRH mRNA, but not GnRH primary transcript (36). It is thus possible that an increase in the rate of posttranscriptional processing during the peripubertal period may lead to increased GnRH production and increased mass of peptide produced and released. The decline in GnRH levels in the adult, however, is not easily explained in this scenario.
Alternatively, the rise in mean GnRH levels in peripubertal animals may be explained by alterations in GnRH proteolytic degradation. The GnRH decapeptide is processed to peptide subproducts, e.g. GnRH (1, 2, 3, 4, 5), primarily through the actions of prolyl endopeptidase and endopeptidase 24.15, and recent work has demonstrated that prolyl endopeptidase activity in particular is decreased during the late juvenile period (37). Thus, an acceleration of GnRH pulsatility during the juvenile-pubertal transition may be unaccompanied by concomitant increases in endogenous peptidase activity, thereby leading to an accumulation of GnRH in the extracellular compartment. Subsequently, the return of mean GnRH levels to lower values in the adult rat could be explained by a possible increase in extracellular degradation due to a return to higher levels of peptidase activity.l}p-g-], 百拇医药
Our observations support the contention that an acceleration of GnRH pulse generator activity functions as the major neural signal for the initiation of puberty in male rats (3), and thus our findings are in direct agreement with those obtained via pulsatile LH measurements in male monkeys (9) and boys (16). It is clear, nevertheless, that there may be major differences in the control of GnRH pulsatility in juvenile rodents compared with primates. The castration of juvenile male monkeys, for example, does not produce a significant increase in GnRH pulse generator activity, whereas in the male rat the same treatment does evoke increased pulsatile LH (and presumably GnRH) secretion. It is thus believed that the GnRH pulse generator in the juvenile monkey is completely or nearly dormant, and hence refractory to the negative feedback actions of gonadal hormones, whereas in the male rat GnRH pulsatility occurs, albeit at low levels, during the late juvenile period. Does this indicate a fundamental difference in the events underlying puberty in the two species? Repeated injection of the GnRH secretagogue, NMA, can induce pubertal maturation prematurely in both monkeys (24) and rats (22), suggesting that changes in GnRH pulse generator activity that drive puberty may differ quantitatively, but perhaps not qualitatively. It is possible that the abbreviated life span of the rat compared with the monkey necessarily compresses the same (or similar) events into a shorter developmental window of time. A dormant period for the GnRH pulse generator may therefore precede the period over which our measurements were made, and the acceleration of GnRH pulse generator activity may commence earlier as well. It is also possible that the GnRH pulse generator is not completely inactive in male primates, as was thought to be the case from early studies in monkeys (9). Indeed, the use of sensitive LH assays in boys has revealed the presence of diminished, yet detectable, pulsatile LH baselines before puberty (16).
The present observations taken together with the previous finding that NMA injections induce precocious puberty (22) suggest that the rate-limiting feature for the pubertal activation of gonadotropin secretion is the electrophysiological activation of GnRH neurons. The molecular and cellular mechanisms that mediate the pubertal activation of the pulse-generating mechanism are not completely understood, but probably depend upon the combined effects of 1) metabolic cues, such as leptin, insulin, GH, IGF, and glucose (38); 2) sensory cues, such as photoperiodic stimuli (39); 3) neuroglial-derived growth factors (40); and/or 4) intrahypothalamic actions of inhibitory and stimulatory neurotransmitters. The mechanisms by which these signals are transduced and integrated to yield an acceleration of GnRH pulsatility remain to be determined.k1elgt/, http://www.100md.com
Acknowledgmentsk1elgt/, http://www.100md.com
We gratefully acknowledge the technical expertise of Ms. Brigitte Mann.k1elgt/, http://www.100md.com
Received July 31, 2002.
Accepted for publication October 3, 2002.s., 百拇医药
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