| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Pediatric Endocrinology |
International Pediatric Growth Research Center, Department of
Pediatrics (K.A.W., S.R., B.L., E.N.), University of Göteborg,
Göteborg; and the Department of Physiology, University of
Ume
(G.S.),
Ume, Sweden; and Children’s Hospital,
University of Helsinki (L.D.), Helsinki, Finland
Address all correspondence and requests for reprints to: Dr. Kerstin Albertsson-Wikland, International Pediatric Growth Research Center, University of Goteborg, Department of Pediatrics, East Hospital, S-416 85 Goteborg, Sweden. E-mail: kerstin.albertsson-wikland{at}pediat.gu.se
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
16 mL). Mean levels of LH
and FSH increased with pubertal development, although the increase in
LH was greater than that in FSH. These increases were due to elevated
basal levels of LH and FSH as well as to increases in the number of
peaks and the peak amplitudes of LH. No diurnal rhythm was found in
boys at stage Pre 1. Thereafter, a clear diurnal rhythm appeared for
LH, and later in puberty, an ultradian rhythm was superimposed, as
shown by time-sequence analyses. A diurnal rhythm also existed for FSH,
but was much less marked than that for LH despite a clear covariation
between LH and FSH, as shown from cross-correlation studies.
Testosterone also showed diurnal variations from the late prepubertal
stage, followed by increasing levels during both day and night in
puberty. We conclude that during puberty, gonadotropin levels rise differently for LH and FSH, which may be due to the development of differences in feedback mechanisms. Despite covariation between LH and FSH, only LH showed a clear diurnal variation. In parallel, nocturnal variations in testosterone and estradiol were found. Changes in mean levels of LH, testosterone, and estradiol as well as their mean daytime and nighttime levels follow each other from the prepubertal stages to late puberty.
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Testosterone secretion follows the same nocturnal pattern as LH during pubertal development (6, 9, 10) and also in young men (11). No such detailed investigations have been performed for estradiol, mainly due to the lack of sensitive methods for determining estradiol concentrations in small volumes. Large and Anderson (12), however, reported no obvious diurnal rhythm of estradiol levels in boys with delayed puberty or in boys with pubertal gynecomastia, even though several individuals had nighttime increases in both testosterone and estradiol in accordance with the studies of Wu et al. (9).
In the present study, 12 healthy boys of normal height were followed semilongitudinally throughout puberty in an attempt to describe the spontaneous pattern of gonadotropin concentration profiles and testosterone/estradiol levels.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Twelve boys were studied at Childrens Hospital (Göteborg,
Sweden). Profiles were taken on 3–9 occasions over a period of 2–9.5
yr, giving a total of 54 profiles (Table 1
). The boys’
chronological ages ranged from 8.7–18.2 yr. All children were healthy
and well nourished, and their heights, weights, and bone ages were
within the normal range. Thyroid, liver, and kidney function tests and
karyotype were normal. Celiac disease was excluded. Puberty was
assessed according to the method of Tanner et al. (13) for
pubic hair and overall genital development and according to the method
of Prader et al. (14) for testicular volume. The boys were
grouped according to testis volume into those at an early pubertal
stage (testicular volume of 4–9 mL), those at the midpubertal stage
(10–15 mL), and those in late puberty (16 mL). The prepubertal boys
were subdivided into 2 groups according to testicular size: boys with
testicular volumes of 1 or 2 mL were referred to as prepubertal 1 (Pre
1), and those with a testicular volume of 3 mL were referred to as
prepubertal 2 (Pre 2). A testicular volume of 3 mL was considered to
reflect the clinical onset of puberty, as these boys already had
clearly higher gonadotropin levels than the boys with smaller
testicular volumes (4). Bone age was always evaluated by the same
radiologist, using the method of Tanner and Whitehouse (TW-2) (15). The
LH and FSH profiles in the early pubertal stages from subjects 1–7 and
10 have been presented previously (4)
|
Assent was obtained from each boy, and informed consent was obtained from his parents. The protocol was approved by the ethical committee of the Medical Faculty, University of Göteborg.
Study protocol
During the first sampling period the children stayed at the hospital for at least 2 days. Subsequently, sampling periods included at least the 24 h of the profile, during which time the boys received a normal diet and were allowed normal activity and sleep. The children’s activity patterns and sleep-wake periods were regularly recorded during all sampling periods.
The following sampling procedure was used for gonadotropin measurement. A heparinized needle (Carmeda, Stockholm, Sweden) was inserted on the first evening or morning, and the collection of blood samples was began between 0800–0900 h. A constant withdrawal pump (Swemed, Goteborg, Sweden) with a nonthrombogenic catheter (Carmeda) was used (16, 17). The rate of withdrawal was 0.5–2 mL/h, and the volume of the testing system was 0.1–0.2 mL. The heparinized tubes were changed every 20 min for 24 h, thus giving 72 samples. The heparinized tubes of blood were stored at room temperature and centrifuged within 24 h. After centrifugation, the plasma samples were frozen and stored until assayed for LH and FSH. Testosterone and estradiol concentrations were measured in samples taken at 1000, 1400, 1800, 2200, 0200, 0400, 0600, and 1000 h; 2.0 mL blood were withdrawn through a venous cannula, and the samples were treated as described for gonadotropins.
Hormone determinations
Gonadotropins. Plasma LH and FSH concentrations were measured by time-resolved immunofluorometric assays, using reagents from Wallac (Turku, Finland), as previously described (3, 4). Each series was stored at room temperature, centrifuged within 24 h, and then analyzed in the same run. The LH standards were calibrated against the WHO International Reference Preparation 68/40, and FSH standards were calibrated against the Second International Reference Preparation of pituitary FSH/LH (78/549). The assay sensitivity for LH was 0.019 IU/L, and that for FSH was 0.014 IU/L, as defined by the mean ±2 SD of 12 replicates of a zero sample. The intraassay coefficient of variation for FSH ranged from 2.1% (at 64 IU/L) to 8.5% (at 0.1 IU/L), and that for LH ranged from 3.1% (at 50 IU/L) to 13.9% (at 0.1 IU/L). The interassay coefficient of variation ranged from 3.6–4.1% for FSH and from 5.4–5.6% for LH at concentrations of about 20 and 5 IU/L, respectively.
Testosterone. Serum concentrations were determined in duplicate by a RIA using coated tube technology (Spectria) from Orion Diagnostics (Espoo, Finland). The volume of the serum used was 50 µL instead of 25 µL to increase the sensitivity of the kit; otherwise, the RIA was conducted according to the manufacturer’s instructions. The detection limit was 30 pmol/L. The intraassay coefficient of variation was 10.6% for 0.21 nmol/L and below 7% for concentrations of 0.42 nmol/L or higher. The interassay coefficient of variation was 31% for 0.19 nmol/L and below 7.4% for concentrations of 0.92 nmol/L or higher.
Estradiol. Serum estradiol concentrations were determined by a RIA using coated tube technology (Spectria) from Orion Diagnostics. The following modifications were made to increase the sensitivity of the kit; 0.7 mL serum was extracted with 4 mL diethyl ether and frozen (-20 C). The ether phase was transferred to tubes, dried under a stream of nitrogen (37 C), and reconstituted in 300 µL zero standard from the kit. From this solution, 150 µL were taken for the RIA, instead of 100 µL, and the incubation period was extended from 2 h to overnight. Otherwise, the RIA was conducted according to the manufacturer’s instructions. The recovery of estradiol using the extraction procedure was 91 ± 5%. The detection limit for the RIA was 6 pmol/L. The interassay coefficient of variation was 27% for concentrations below 15 pmol/L, and 17% for concentrations between 15–30 pmol/L. The intraassay coefficient of variation was 16% for concentrations below 15 pmol/L, 14% for concentrations between 15–25 pmol/L, 12% for concentrations between 25–50 pmol/L, and 8% for concentrations between 50–100 pmol/L.
Analysis of the 24-h profiles
Pulse detection. LH and FSH pulse analyses were performed using a computerized pulse analysis program, the Pulsar program developed by Merriam and Wachter (18). The program identifies peaks by height and duration from a smoothed baseline, using the assay SD as a scale factor. The cut-off parameters G1 to G5 of the Pulsar program were set at 2.5, 1.5, 1, 0.75, and 0.5 times the intra-assay SD as criteria for accepting peaks 1, 2, 3, 4, and 5 points wide, respectively, and the peak-splitting parameter was set at 1.5. With these settings, the program did not detect any peaks when 72 consecutive samples from each of 2 different plasma pools were assayed. Missing values comprised less than 3% of the total samples and were not included in the calculations.
Fourier time-series analysis for diurnal variation. The original hormone concentration-time series was smoothed with a three-point moving average (weights w1 = w2 = 1/4; w0 = 1/2) to reduce the influence of high frequency components. The smoothed series was analyzed as Fourier expansions (i.e. absolute spectral power) (19). Spectral analysis provides different information from that provided by pulse-counting techniques. The regular oscillatory components of a pulsatile profile were analyzed; a profile with many randomly occurring pulses would have a flat transform. The Fourier analysis was made on the original data sets and also on data sets made stationary (stationarized) before time-series analysis. Stationarizing data sets effectively removed the long term trends, making it easier to display the higher frequency components
Cross-correlation. Cross-correlation was used to analyze the relationships between LH and FSH. This is a technique for assessing the time relationship between two data series. The stationarized data were progressively moved at intervals corresponding to the sampling interval, and this lag-time was varied between -3 to +3 h. The data were regressed with each other at each move, and a correlation coefficient was generated for each point between LH and FSH (20, 21, 22).
Statistical analyses
Data are given as the mean ± SEM. Data from
subjects with more than one observation period per pubertal stage were
averaged (at each time point) in the graphs and for statistical
analysis. Differences between the groups were assessed by the
Mann-Whitney U test. P < 0.05 was considered
significant. The distribution of peak amplitudes between pubertal
stages was compared by 
2 test.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Figure 1 shows LH and FSH profiles and their
cross-correlations for a representative individual (subject 2). Mean
(±SEM) levels of LH and FSH increased with pubertal
stages; the LH levels increased up to midpuberty, and FSH levels
continued to increase into late puberty (Fig. 2).
Cross-correlation between LH and FSH levels was also evident and was
most pronounced in the later stages of puberty (Fig. 2).
|
|
Fourier analyses revealed distinct diurnal rhythms in the
LH profiles for all prepubertal and pubertal stages except in the
earliest prepubertal stage, Pre 1 (Fig. 3). Furthermore,
with advances in pubertal maturation, higher frequency components for
LH emerged, visually enhanced in the stationarized datasets, with
dominating periods of 90–180 min (Fig. 3). There were also diurnal
rhythms in FSH levels during the early, mid-, and late pubertal stages,
but to a much lesser degree than for LH.
|
LH. There was a marked increase in all the parameters of LH
levels (mean level, maximum level, baseline, number of peaks, and mean
peak amplitude) during the initial phase of pubertal progression (Table 2). The baseline levels of LH increased by about 15
times from the Pre 1 stage up to midpuberty, whereas the number of
peaks increased almost 3-fold during the same period (Table 2). There
was also a marked progressive shift in the peak amplitudes of LH during
pubertal development from being mainly below 0.5 mU/L (>80% of the
peaks) at the Pre 1 stage to being mainly above 1 mU/L (>80% of the
peaks) at the late pubertal stage, as indicated in Fig. 4.
|
|
6-fold) from Pre 1 to late puberty. In contrast to LH, neither the
number of peaks nor the peak amplitude of FSH showed a distinct
increase with pubertal development (Table 2
and Fig. 4).
Figure 5 shows all estimates of number of peaks, mean
values, and baseline values for FSH and LH in all 12 normal boys, where
all longitudinal points from the same subject are connected with
lines.
|
The mean testosterone concentration rose with pubertal development
(Fig. 6 and Table 3). Figure 7 shows testosterone and estradiol levels for one
representative individual (subject 2). In general, the concentrations
of both testosterone and LH were low during the day and high at night,
although completely irregular pulsatile patterns occurred in
approximately 5% of the profiles. When evaluating individual curves, a
steady nadir was observed between 1200–2400 h. However, in at least
one curve from each boy, testosterone levels were still falling at
1300 h, and the nocturnal rise was sometimes evident at 2400
h. Thus, we restricted the day period to 4 h, that is between
1800–2200 h, for testosterone, estradiol, and LH levels. In a similar
fashion, the night period was restricted to between 0200–0600 h (Table 3). Daytime levels of testosterone did not change significantly during
the prepubertal period (Pre 1 and Pre 2), but rose continuously
throughout the whole pubertal period, whereas LH levels rose mainly up
to midpuberty, as discussed above (Table 3). Nighttime levels of
testosterone increased significantly in early puberty (Table 3) and
continued to increase throughout puberty.
|
|
|
).
Estradiol levels were below the detection limit during the Pre 1 and
Pre 2 stages and began to rise, mainly during the night, in some of the
boys in early puberty. At mid and late puberty, the 24-h pattern of
estradiol was remarkably similar to that of testosterone (Fig. 5 and
Table 3). There was a clear correlation between testosterone and
estradiol during mid (r = 0.70; P < 0.0001;
n = 79) and late (r = 0.70; P < 0.0001;
n = 73) puberty.
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Clinical and experimental studies have highlighted the role of GnRH in the regulation of LH and FSH secretion, and have indicated that LH secretion is much more dependent on GnRH than is FSH secretion (1, 25). In sheep, monkeys, and humans, a GnRH pulse frequency of one pulse per h induces the release of both LH and FSH, whereas a lower GnRH pulse frequency of one pulse every 3–4 h maintains FSH secretion only, and LH secretion declines (25, 26, 27). Gonadotropin secretion in prepubertal boys with testicular volumes of 1–2 mL is consistent with low GnRH pulse frequencies and low LH levels compared with those of FSH. At this stage, the central inhibition of GnRH secretion is probably maximal. A dramatic change in GnRH pulse generator activity probably occurs during the development from a prepubertal stage with a testicular volume of 1–2 mL to the prepubertal stage with a testicular volume of 3 mL, as over this time LH levels increase 10-fold during the night and the pulse frequency is doubled. This marks the end of the period of GnRH pulse generator inhibition and starts the process of puberty. However, it is not until a testicular volume of 4–9 mL is reached that a substantial increase in testosterone levels is seen, which enables sexual maturation to progress into early puberty.
The present study emphasizes another intrinsic property of the GnRH pulse generator; that is, the diurnal rhythm, which is apparent in males after adolescence (11, 28). All boys, except those who were prepubertal with a testicular volume of 1–2 mL, had a diurnal rhythm of LH, as determined by Fourier analysis and the day/night ratio. Again, the less GnRH pulse-dependent FSH has a diurnal rhythm with a lower spectral power than that of LH. The factors controlling FSH secretion are, however, less clear than those controlling LH secretion due to several factors. For example, the dependency of FSH secretion on GnRH decreases during puberty (29), it is influenced not only by testosterone/estradiol but probably also by inhibin from the testis (1, 24), and it has a longer half-life (180–200 min) than LH (60–90 min) (1). The most dramatic increase in FSH levels takes place during development from a testicular volume of 1–2 mL (Pre 1) to 3 mL (Pre 2), which is a period when the secretion of both testosterone and inhibin is low (30). FSH levels increased, however, steadily up to midpuberty. Despite the different developmental patterns of LH and FSH levels seen during puberty, the release of FSH and LH occurred simultaneously, as determined by cross-correlation analysis. The longer half-life of FSH can partly explain this phenomenon (1). The increase in LH dependency and decrease in FSH dependency on GnRH, previously observed during puberty in girls (29) and adult women (31), is also apparent in these boys.
The intrinsic diurnal rhythm of the GnRH pulse generator is also obvious in the nocturnal levels of testosterone; all boys except those who were prepubertal, with a testicular volume of 1–2 mL, had higher levels of testosterone during the night. Estradiol levels also followed this pattern, although to a lesser degree, with a diurnal pattern in midpuberty. The synchronization of estradiol and testosterone levels probably represents aromatization of testicular testosterone to estradiol, although the amount of testosterone aromatized in the testis and peripheral tissues is unknown during pubertal development. However, in young men, testosterone is secreted in pulses, and estradiol levels in testicular venous blood samples follow a similar pattern. These pulses of estradiol are, however, not as evident as those in peripheral blood (32). The lack of a diurnal pattern of estradiol in late puberty is probably a reflection of increasing levels during the day as well as a limited aromatizing capacity. From a practical clinical point of view, Wu et al. (33) proposed that early morning testosterone measurements reflect the nocturnal rise; this is confirmed by the results of the present study.
In conclusion, this study shows that very specific changes occur in FSH, LH, testosterone, and estradiol levels during pubertal development in boys, and that there is a synchronization of LH and FSH concentration profiles. FSH levels increase up to early puberty and exhibit only a moderate diurnal rhythmicity. Levels of LH, in contrast, are very low before puberty and rapidly develop a marked diurnal rhythm, with high nighttime levels, on which are superimposed ultradian rhythms. The 24-h patterns of testosterone and estradiol levels closely follow those of LH. Thus, to register the early hormonal changes that reflect pubertal development, nighttime levels of LH and/or testosterone have to be measured.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received March 18, 1996.
Revised October 22, 1996.
Accepted October 28, 1996.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
-subunit secretion from the
gonadotrope by gonadotropin-releasing hormone (GnRH): evidence from the
use of two GnRH antagonists. J Clin Endocrinol Metab. 70:328–335.[Abstract]
This article has been cited by other articles:
 |
|
 |
|
  K. Allvin, C. Ankarberg-Lindgren, H. Fors, and J. Dahlgren Elevated Serum Levels of Estradiol, Dihydrotestosterone, and Inhibin B in Adult Males Born Small for Gestational Age J. Clin. Endocrinol. Metab., April 1, 2008; 93(4): 1464 - 1469. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. Chagin and L. Savendahl GPR30 Estrogen Receptor Expression in the Growth Plate Declines as Puberty Progresses J. Clin. Endocrinol. Metab., December 1, 2007; 92(12): 4873 - 4877. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-J. He, M. Hirata, N. Yamauchi, S. Hashimoto, and M.-a. Hattori The disruption of circadian clockwork in differentiating cells from rat reproductive tissues as identified by in vitro real-time monitoring system J. Endocrinol., June 1, 2007; 193(3): 413 - 420. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Aksglaede, A. Juul, H. Leffers, N. E. Skakkebaek, and A.-M. Andersson The sensitivity of the child to sex steroids: possible impact of exogenous estrogens Hum. Reprod. Update, July 1, 2006; 12(4): 341 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Hero, E. Norjavaara, and L. Dunkel Inhibition of Estrogen Biosynthesis with a Potent Aromatase Inhibitor Increases Predicted Adult Height in Boys with Idiopathic Short Stature: A Randomized Controlled Trial J. Clin. Endocrinol. Metab., December 1, 2005; 90(12): 6396 - 6402. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S Ramaswamy, C R Pohl, G R Marshall, and T M Plant A switch from continuous to episodic testicular testosterone release in response to pulsatile LH stimulation in juvenile rhesus monkeys (Macaca mulatta) J. Endocrinol., October 1, 2004; 183(1): 61 - 68. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.D. Alvarez, D. Chen, E. Storer, and A. Sehgal Non-cyclic and Developmental Stage-Specific Expression of Circadian Clock Proteins During Murine Spermatogenesis Biol Reprod, July 1, 2003; 69(1): 81 - 91. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T Hanaoka, N Kawamura, K Hara, and S Tsugane Urinary bisphenol A and plasma hormone concentrations in male workers exposed to bisphenol A diglycidyl ether and mixed organic solvents Occup. Environ. Med., September 1, 2002; 59(9): 625 - 628. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Plant and G. R. Marshall The Functional Significance of FSH in Spermatogenesis and the Control of Its Secretion in Male Primates Endocr. Rev., December 1, 2001; 22(6): 764 - 786. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ramaswamy, T. M. Plant, and G. R. Marshall Pulsatile Stimulation with Recombinant Single Chain Human Luteinizing Hormone Elicits Precocious Sertoli Cell Proliferation in the Juvenile Male Rhesus Monkey (Macaca mulatta) Biol Reprod, July 1, 2000; 63(1): 82 - 88. [Abstract] [Full Text]
|
|
|
|
|
|
A. P. Cemeroglu, G. B. Kletter, W. Guo, M. B. Brown, R. P. Kelch, J. C. Marshall, V. Padmanabhan, and C. M. Foster In Pubertal Girls, Naloxone Fails to Reverse the Suppression of Luteinizing Hormone Secretion by Estradiol J. Clin. Endocrinol. Metab., October 1, 1998; 83(10): 3501 - 3506. [Abstract] [Full Text]
|
|
|
|
|
|
D. J. Phillips, K. Albertsson-Wikland, K. Eriksson, and L. Wide Changes In the Isoforms of Luteinizing Hormone and Follicle-Stimulating Hormone during Puberty In Normal Children J. Clin. Endocrinol. Metab., September 1, 1997; 82(9): 3103 - 3106. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
