Hypophyso-gonadal function in humans during the first year of life. 1. Evidence for testicular activity in early infancy.

Total and unbound testosterone and Delta(4)-androstenedione have been determined in 104 cord blood samples. The same sexual steroids and pituitary gonadotropins have been measured in 46 normal male infants aged 27-348 days and 34 normal female infants aged 19-332 days. In cord blood of female neonates mean total and unbound testosterone was 29.6+/-7.5 and 0.89+/-0.4 ng/100 ml, respectively (mean+/-1 SD); Delta(4)-androstenedione was 93+/-38 ng/100 ml. In male neonates mean plasma total and unbound testosterone was 38.9+/-10.8 and 1.12+/-0.4 ng/100 ml; Delta(4)-androstenedione was 85+/-27 ng/100 ml. In female infants testosterone concentrations remained constant during the 1st yr of life with a mean concentration of 7+/-3 ng/100 ml. Mean unbound testosterone and Delta(4)-androstenedione concentrations were 0.05+/-0.03 and 16.7+/-8.3 ng/100 ml, respectively. Mean plasma levels of follicle-stimulating hormone and luteinizing hormone were 8.7+/-3.3 and 12.9+/-7.7 mU/ml. In male infants mean plasma total testosterone concentration increased to 208+/-68 ng/100 ml from birth to 1-3 mo of age, decreasing thereafter to 95+/-53 ng/100 ml at 3-5 mo, 23.2+/-18 ng/100 ml at 5-7 mo, and reached prepubertal levels (6.6+/-4.6 ng/100 ml) at 7-12 mo. Mean unbound testosterone concentration plateaued from birth to 1-3 mo of age (1.3+/-0.2 ng/100 ml) decreasing to prepubertal values very rapidly. Mean Delta(4)-androstenedione concentration, although progressively decreasing during the 1st yr of life to 11.7+/-4.5 ng/100 ml, was higher than in the female at 1-3 mo of life (34+/-11 ng/100 ml). Mean plasma level of follicle-stimulating hormone was 6.7+/-2.9 mU/ml, and that of luteinizing hormone was 19.7+/-13.5 mU/ml, significantly higher than in the female. There was no correlation between gonadotropin and age or testosterone. The present data demonstrate that the testes are active during the first natal period. It is tempting to correlate this phenomenon to a progressive maturation of the hypothalamo-pituitary-gonadal axis. It is possible that the surge in testosterone occurring the first 3 mo could play a role in the future life pattern of the male human being.

Daily administration of melatonin delays rat vaginal opening and disrupts the first estrous cycles: evidence that these effects are synchronized by the onset of light.

The effect of daily melatonin administration was investigated in the immature female rat. Starting on day 15 of age, 100 micrograms melatonin were injected sc at different times of the day in animals housed in 12 h of light, 12 h of darkness or 16 h of light, 8 h of darkness. Melatonin given 9-11 h after the onset of light in both lighting regimens resulted in a 10-day delay of vaginal opening, a dissociation of the relation between vaginal opening and first proestrus, and a disruption of the initial estrous cycles. The same dose of melatonin given at other times during the photoperiod had no effect on sexual maturation. GnRH secretion in melatonin-treated animals was decreased, as judged by 30% lower pituitary GnRH receptor number in animals killed after opening of the vagina. During the diestrous phases, plasma levels of LH, FSH, and 17 beta-estradiol were similar to those in control rats, but during proestrus, the surge of FSH was higher, and the peak of estradiol was higher and of a longer duration. This hormonal pattern suggests a build-up of hormones in secreting cells, which follows the lower incidence of proestrous phases in melatonin-treated rats. This build-up of FSH was indeed present, with higher concentrations in the pituitary during diestrus after melatonin treatment, while pituitaries removed during proestrus had lower contents of FSH. These results confirm that chronic melatonin administration delays sexual maturation of female rat, probably by retarding maturation of hypothalamic GnRH-producing cells. Thus, melatonin could modify basal GnRH secretion or pulsatile release. Pituitary and ovarian responsiveness do not seem to be affected, since proestrous surges of 17 beta-estradiol, LH, and FSH occur, albeit at a reduced frequency. The results also show that there is a window of maximum sensitivity to administration of melatonin 9-11 h after the onset of light, and that this window of sensitivity is synchronized by the onset of light. This raises the possibility that the abnormal presence of endogenous melatonin during this period of the day could induce abnormal sexual development.

Binding kinetics of the long-acting gonadotropin-releasing hormone (GnRH) antagonist antide to rat pituitary GnRH receptors.

The GnRH antagonist Antide has been shown to produce prolonged inhibition of gonadotropin secretion in ovariectomized monkeys and other animal models. The reasons for such a long duration of action have not yet been clarified. To understand the mode of action of this new antagonist, we have performed association and dissociation binding kinetics using either crude rat pituitary homogenates as source of GnRH receptors or dispersed pituitary cells in culture. The binding characteristics of the radioiodinated Antide analog 125I-labeled[D-Tyr0] Antide to GnRH receptors in rat pituitary homogenates were comparable to those of the first generation GnRH antagonist 125I-labeled [Ac(3)Pro1,pFD-Phe2,D-Trp3,6]GnRH or the GnRH agonist 125I-labeled [D-Trp6,(N-Et)Pro9,Des,Gly10]GnRH, with an affinity constant (Ka) in the 10(10) M-1 range. The maximum binding capacity was consistently higher with the antagonist tracers than with the [125I]GnRH agonist. Both antagonists dissociated at a slower rate at 4 C (approximately 4 times) than the [125I]GnRH agonist. Incubation at 23 C of 125I-labeled [D-Tyr0] Antide previously bound at 4 C resulted in complete dissociation within 8 h after the addition of an excess amount of any of the GnRH analogs; in addition, simple dilution of the incubation medium produced spontaneous dissociation at this temperature. Using rat pituitary cells, Antide was found to inhibit the LH response to native GnRH (10(-8) M) in a dose-related manner. To test whether the binding of Antide is normally reversible at 37 C, Antide (10(-7) M) was added to the culture medium 3 days after cell plating, and the initial preincubation was resumed for 24 h. Cells were then washed twice, and dissociation was allowed to take place. Bound Antide was shown to dissociate rapidly at 37 C, as cells previously treated with Antide produced a full LH response within 24 h if challenged with native GnRH. In conclusion, the binding kinetics of 125I-labeled [D-Tyr0]Antide to GnRH receptors, which should reflect those of Antide, did not present abnormal features. Although this antagonist, similar to other GnRH antagonists, dissociated from pituitary receptors at a slower rate than GnRH analogs, rapid and spontaneous dissociation was achieved at 23 C with simple dilution, and dissociation of unmodified Antide occurred at 37 C. Taken together, our results support the concept that the long duration of action of Antide is not due to any toxic effect of Antide at the receptor site and could derive only marginally from the slow dissociation rate of this antagonist.

Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor delays sexual maturation in the male rat.

GH deprivation after passive immunization against rat GRF (rGRF) markedly affects somatic growth in male rats. Since it has been postulated that GH and probably insulin-like growth factor-I (IGF-I) might have a permissive role on sexual maturation, the effects of GH deprivation on the course of sexual maturation were tested. Male rats were treated with a potent anti-rGRF serum between 15 and 39 days of life (0.25 ml administered sc every second day). Body weight of treated rats averaged 62% of that of control (normal rabbit serum-treated) rats at 40 days of life (d), and 64% at 50 d after which age, treated rats started to grow normally. At 40 and 50 d, pituitary GH content was very much depressed (representing approximately 20% of control values at both ages), plasma GH was undetectable, and plasma IGF-I levels averaged 30% of those of control rats. At 70 d, 30 days after cessation of treatment, pituitary GH content, and IGF-I secretion were almost normal. At 40 d, testes and seminal vesicles of treated rats were small-for-age in agreement with significantly decreased plasma levels of FSH and delayed spermatogenesis characterized by the presence of only few or no spermatozoa. At 50 d, 10 days after cessation of anti-rGRF injections, progress of sexual maturation was found to be consistent with age and coincided with normalization of growth rate. At 40 and 50 d, pituitary contents of FSH and LH were severely decreased but became normal at 70 d. In conclusion, GH deprivation which markedly affected somatic growth induced a transient delay of sexual maturation. GH deficiency seems to have affected mostly the synthesis and secretion of FSH, thus producing a delay in testes growth and in the differentiation of the germinal cells. The low levels of IGF-I might also have been the cause for the delay of maturation at the pituitary and/or the gonadal levels.

Plasma growth hormone (GH) response to intravenous GH-releasing factor (GRF) in adult rats: evidence for transient pituitary desensitization after GRF stimulation.

The ability of human (h)GRF-(1-29)NH2 to stimulate GH secretion was studied in cannulated adult rats. In order to suppress endogenous GRF secretion and the inhibitory action of hypothalamic somatostatin (SRIF), rats were anesthetized with sodium pentobarbital. Intravenous administration of hGRF-(1-29)NH2 elicited a dose-dependent response of plasma GH, with 250 ng/kg being the smallest effective dose in male rats. In female rats, for each dose tested (250 to 70,000 ng/kg), the GH response represented only about 60% that of male rats. Repeated iv stimulations with hGRF-(1-29)NH2 at short time intervals (45 min) produced transient desensitization of pituitary responsiveness to GRF: a blunted GH response to the second and third stimulations was observed both in male and in female rats and for each dose tested. Similar blunted responses were also obtained with repeated injections of native hGRF-(1-44)NH2. The possibility that these blunted responses could be due to incomplete suppression of hypothalamic SRIF secretion by sodium pentobarbital was excluded by the use of rats that were passively immunized against SRIF; in these rats, it was shown that at least 65% of the inhibition of the GH response after the second GRF stimulation was unrelated to SRIF action. Similar transient desensitization to repeated hGRF-(1-29)NH2 stimulations was also observed in conscious rats that were passively immunized against SRIF. This occurrence of blunted responses was shown to be related to the length of the time interval between GRF stimulations, with longer intervals resulting in less or no desensitization. It appears thus that modulation of pituitary responsiveness to the action of GRF is mediated by at least two independent mechanisms in the rat: in addition to the inhibitory action imposed by hypothalamic SRIF, which induces periods of refractoriness to the action of GRF, it was shown in this study that in the pituitary level each GRF stimulation also induces a transient desensitization of somatotrophs for about 1 h. This period of refractoriness might not be due to excessive stimulation with GRF, since it was also observed with the lowest dose of hGRF-(1-29)NH2 that gave a significant release of GH. Finally, a sex difference was confirmed for the response of anesthetized adult rats to stimulation with hGRF-(1-29)NH2, reflecting a sex steroid-induced modification of pituitary responsiveness to GRF stimulation.

Evidence that neuropeptide Y could represent a neuroendocrine inhibitor of sexual maturation in unfavorable metabolic conditions in the rat.

Neuropeptide Y (NPY) is known to be involved in the central regulation of appetite, sexual behavior and reproductive function. Whereas central administration of NPY strongly stimulates feeding, diet restriction produces overexpression of NPY in the arcuate and paraventricular nuclei that might reflect behavioral adaptations to shortage of food. The role of NPY for the regulation of sexual function is still controversial. Whereas NPY is stimulatory during proestrus in the rat, acute administration of NPY is inhibitory in castrated animals and we have shown that chronic administration of NPY inhibits both the gonadotropic and somatotropic axis in adult female rats. In order to further analyse the role of NPY during sexual maturation, a model of delayed sexual maturation imposed by food restriction and return to ad-libitum feeding was used. Young female rats were restricted to 7-8 g food daily starting at 24 days of life (d). This restriction completely prevented sexual maturation. At 50 d, ICV cannulas were placed and at 60 d, Alzet minipumps either delivering NPY (18 micrograms/day) or vehicle into the ICV cannula were implanted dorsally. At 61 d, rats were switched to ad-libitum feeding, a change that produced vaginal opening within 4 days in all vehicle-treated rats. In the rats receiving NPY, significantly increased food intake and weight gain were observed but only one out of the 9 rats studied experienced vaginal opening at 66 d, the other 8 animals remaining sexually immature at 67 d at sacrifice. Sexual immaturity of NPY-treated rats was further confirmed by decreased ovarian weight and reduced number of pituitary GnRH receptors. Plasma IGF-I levels were markedly reduced in NPY-treated rats. Since food restriction has been shown both to increase hypothalamic NPY and to reduce or inhibit sexual function, these data bring evidence for the first time that NPY could be involved in the inhibition of sexual maturation imposed by food restriction, since maintenance of elevated NPY levels in the hypothalamus did prolong this state of sexual immaturity despite restoration of normal food intake.

Growth hormone (GH) deprivation induced by passive immunization against rat GH-releasing factor does not disturb the course of sexual maturation and fertility in the female rat.

The importance of normal GH secretion for the onset of sexual maturation is a subject of controversy. Also, the need to achieve a minimal body size or body fat content has been postulated to be of importance for determining the timing of the onset of puberty. To evaluate the importance of GH secretion on the onset of sexual maturation in the female rat, GH deprivation has been induced by treating prepubertal rats with antirat GRF serum to passively immunize these animals against GRF. Chronic administration of anti-GRF serum produced in all series an impressive reduction in growth rate (from 5 to 2 g/day), resulting in a body weight averaging 50-60% the normal value at 50 days of life. Despite this deficit in growth, sexual maturation, as established by vaginal opening and first estrous cycles, occurred at the normal age in three of four series of rats; in one series, however, sexual maturation was delayed by 4 days, but thereafter, all parameters indicated that the gonadotropic axis was normally activated. In one series, fertility was tested at 59 days of age in females with a body weight corresponding to 51% of the control weight; these females conceived and delivered a reduced number of pups (9.4 +/- 0.7 instead of 14.2 +/- 0.8 in control dams), but the pups were of normal size. In a second experimental approach, the effect of GH deprivation was evaluated in a model of late sexual maturation obtained by severe food restriction followed by a switch to ad libitum feeding. Severe food restriction initiated at approximately 28 days, when the body weight was 75 g, drastically reduced the growth rate and completely prevented sexual maturation. A switch to ad libitum feeding at 50 days provoked an important compensatory growth and the occurrence of sexual maturation 4 days later. Passive immunization against GRF during this recovery phase did reduce the growth rate, but did not delay sexual maturation. Plasma insulin-like growth factor-I (IGF-I) secretion was very low in food-restricted rats and in each situation with induced GH deprivation. During food restriction, plasma IGF-binding protein-3 (IGFBP-3) and to a lesser extent IGFBP-1 were decreased, and IGFBP-2 was increased; after switching to ad libitum feeding, plasma levels of IGFBP-2 normalized, but levels of IGFBP-1 and IGFBP-3 remained low in the face of normalized plasma IGF-I levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Ontogeny of hypothalamic luteinizing hormone-releasing hormone (GnRH) and pituitary GnRH receptors in fetal and neonatal rats.

Although it is known that LH secretion starts at 17 days of gestation in the fetal rat and that this first LH release is most likely driven by hypothalamic GnRH, an earlier role for GnRH during fetal life has been postulated with the observation that presence of GnRH is important before day 13 of gestation for the differentiation of the pituitary anlage. In order to clarify the different roles of GnRH during fetal life, we have studied the first appearance of GnRH in the fetal brain, the expression of GnRH receptors in the fetal pituitary gland, and the presence of GnRH immunoreactivity within the fetal gonadotrophs. GnRH was present in the earliest brain tissue examined (12 days of gestation). From 12-17 days, GnRH content of fetal brain remained low and then increased markedly by the end of gestation. No immunoreactive GnRH-like material could be detected in rat placental tissue throughout gestation. Binding sites for GnRH were detected as early as 12 days of gestation in fetal pituitary glands. However, binding was very low until 16 days. At 17 days, Scatchard analysis indicated the presence of high affinity, low capacity binding sites [affinity constant (Ka) = 10(10) M-1]. Intracellular presence of GnRH as seen by immunocytochemistry using ultrathin sections prepared by cryoultramicrotomy was first visible at 14 days and started to increase at 16 days. LH was first detectable in the fetal pituitary by RIA at 17 days; FSH was first detectable at 21 days, and PRL at 1 day of postnatal life. Thereafter, neonatal pituitary contents of LH, FSH, and PRL increased linearly with-time, as did the number of pituitary GnRH receptors. At 10 days of postnatal life, pituitary contents of LH and FSH were significantly higher in females than in males. In summary, hypothalamic GnRH appears early in fetal life and potentially can induce differentiation of the pituitary anlage. Conversely, the presence at 15 days of gestation of specific binding sites for GnRH and of intracellular GnRH immunoreactivity in gonadotrophs indicates that the hypophysiotropic action of GnRH clearly precedes the start of LH biosynthesis.

Daily afternoon administration of melatonin does not irreversibly inhibit sexual maturation in the male rat.

Previous studies from our laboratory demonstrated that daily afternoon melatonin injections from 20-40 days of age inhibited sexual development of young male rats, whereas in adult animals, similar injections had no effect. The present study was designed to determine more precisely the critical age period during which melatonin exerts its inhibiting effect and to see whether spontaneous sexual maturation resumes after discontinuation of melatonin administration at 45 days of age or even during continuous administration of melatonin until 115 days of age. Sexual maturation was evaluated using weights of seminal vesicles and testes; plasma levels of testosterone, FSH, and LH; pituitary contents and concentrations of FSH and LH; and, finally, pituitary content of GnRH receptors. Administration of melatonin to young male rats from 20-30 days of life had the same inhibitory effect on sexual maturation at 40 days as melatonin injections from 20-40 days. In contrast, administration of melatonin from 30-40 days only slightly decreased plasma testosterone concentration, weight of seminal vesicles, and pituitary GnRH receptor content. Melatonin administration from 38-40 days had no effect. Daily melatonin administration from 20-45 days of age was followed by resumption of sexual maturation, as observed at 70 days. The recovery was complete by 80 days of age when all of the parameters studied reflected complete sexual maturation. Finally, in rats treated continuously with melatonin from days 20 until 115, sexual maturation occurred but was delayed by about 20-30 days. Beginning of sexual development was observed at 60 days of life, and full development was attained only at 100 days. These data confirm that melatonin delays sexual maturation in the young male rat when administered daily in the afternoon. They demonstrate that this inhibitory action of melatonin is most critical between 20 and 30 days of life and is reversible regardless of whether melatonin administration is discontinued after 45 days of life. The suppression of the pubertal peaks of pituitary GnRH receptor number and pituitary and plasma FSH concentrations in treated rats suggests that melatonin interferes with the pubertal increase in GnRH secretion. In conclusion, these reversible effects of melatonin suggest that this pineal indolamine represents an important factor for the timing of sexual maturation.

Influence of exogenous melatonin on melatonin secretion and the neuroendocrine reproductive axis of intact male rats during sexual maturation.

The influence of daily sc administration of melatonin (5-100 micrograms/day) on sexual development of prepubertal and pubertal male rats was studied in vivo. Adult animals were also studied. When melatonin was injected daily into young animals starting at day 20 of age, dose-dependent reductions in plasma testosterone, testis and seminal vesicles weights, plasma FSH and LH levels, and pituitary GnRH receptor number were observed at day 40 or 45 of age when animals were killed. In contrast, prepubertal animals (5-20 days old) showed no significant responses to similar treatment with melatonin, whereas in adult animals (70-90 days old), melatonin elicited only a small decrease in plasma testosterone concentration. Melatonin analogs such as N-acetyl-serotonin and 5-hydroxytryptophol administered daily from 20-45 days of age did not produce any effect. Chronic melatonin administration from 20-50 days of life did not alter the occurrence of the nocturnal rise of circulating plasma melatonin, but did enhance its amplitude. Our results demonstrate that exogenous melatonin can inhibit or delay sexual maturation in the male rat if administered between 20 and 40 days of age and suggest that this inhibitory action is exerted at the hypothalamic and/or pituitary level.

Pituitary receptor sites for gonadotropin-releasing hormone: effect of castration and substitutive therapy with sex steroids in the male rat.

To examine the role of pituitary gonadotropin-releasing hormone (GnRH) receptors (pit GnRH-R) in the regulation of gonadotropin secretion, male rats were orchidectomized and then selectively received substitutive therapy with sex steroids. Pituitary content of GnRH-R was determined by saturation analysis, using radioiodinated [D-Trp6,(N-Et)Pro5,des-Gly10]GnRH as tracer. Castration produced a rapid and sustained increase of the number of GnRH-R, which doubled after 2 days, and after 10 days the pituitary content of GnRH-R was 258 +/- 23 fmol/pituitary compared to 103 +/- 12 fmol/pituitary for sham-operated control animals. No change of the affinity constant (Ka) was observed (Ka = 1.13 +/- 0.08 X 10(10) M-1; n = 14). Plasma LH increased 5- to 10-fold and FSH-2- to 3-fold after castration, and hypothalamic GnRH content was depleted by 30-60%. Immediate substitution of castrated rats with testosterone propionate (250 micrograms daily) prevented the increases of both plasma gonadotropins and of GnRH-R. Treatment of acutely castrated rats for 7 days with testosterone propionate (50-200 micrograms), 5 alpha-dihydrotestosterone propionate (25-400 micrograms), or estradiol benzoate (2 micrograms) prevented the rise in pit GnRH-R in a dose-related manner and normalized the other parameters studied except that plasma FSH remained slightly elevated. In contrast, when substitutive therapy was started 8 days after castration or later, the 7-day treatment with sex steroids reduced plasma gonadotropins, but pit GnRH-R remained elevated, and hypothalamic GnRH content remained depleted. These results indicate that the marked increase of gonadotropin secretion after castration is mediated at least in part, by an increase in the number of pit GnRH-R. Sex steroids were able to reverse all castration-induced endocrine changes in acutely castrated rats, but in long term castrated animals their action at higher centers to normalize hypothalamic GnRH content, and indirectly, to reduce pit GnRH-R content, was either delayed or ineffective. Thus, the rapid feedback action of sex steroids in long term castrated rats may be predominantly exerted at the pituitary level.

Neuropeptide Y administered chronically into the lateral ventricle profoundly inhibits both the gonadotropic and the somatotropic axis in intact adult female rats.

Neuropeptide Y (NPY) is known to be involved in the central regulation of appetite, sexual behavior, and reproductive functions. Whereas central administration of NPY strongly stimulates feeding in satiated animals, diet restriction produces overexpression of NPY in the arcuate and paraventricular nuclei that might reflect behavioral adaptations to shortage of food. Previous studies indicated that central administration of NPY resulted in controversial actions on LH secretion, either stimulatory or inhibitory. In order to analyze the chronic effect on pituitary function of centrally administered NPY, stainless steel cannulae were implanted in the right lateral ventricles of intact 45-day-old Sprague-Dawley female rats. Ten days later, Alzet osmotic minipumps filled with saline or different concentrations of NPY adjusted to deliver 3, 6, 12, or 18 micrograms/day were connected to the intracerebroventricular (icv) cannulae, implanted sc dorsally, and the effects of these treatments evaluated after 7 days. Chronic icv infusion of NPY produced the expected dose-related increase in food intake [25.3 +/- 0.8 g/day (basal) to 47.9 +/- 4.3 g/day (highest NPY dose)] and body wt gain (3.7 +/- 0.4-11.5 +/- 1.4 g/day). Basal insulinemia was highly correlated to the increase in food intake. This orexigenic action of NPY was accompanied by a drastic dose-related decrease in pituitary wt (14.0 +/- 0.5-8.3 +/- 0.3 mg), pituitary concentration of GnRH receptors, a known marker of the activity of the hypothalamo-pituitary gonadal axis (15.2 +/- 1.7-5.2 +/- 0.5 fmol/mg), and ovarian wt (84.0 +/- 4.2-49 +/- 6.7 mg). Ovulation was impaired in NPY-treated animals as seen by daily inspection of vaginal smears. A sharp dose-dependent decrease in plasma levels of insulin-like growth factor I was also observed [934 +/- 64 ng/ml (basal) to 385 +/- 26 ng/ml (highest NPY dose)], probably secondary to a decrease in GH secretion. Whereas these data confirm the central action of NPY to stimulate appetite in satiated animals, they provide the first demonstration that chronic icv administration of NPY unequivocally inhibits gonadotropin secretion and sexual function in intact female rats. These data also confirm that NPY can suppress GH secretion and other anabolic hormones. In conclusion, these results may indicate a physiological role of NPY as an integrator of different adaptive behaviors in periods of unfavorable metabolic conditions such as diet restriction, extending its action to inhibition of sexual functions and anabolic processes.

Failure of dehydroepiandrosterone enanthate to promote growth.

Adrenal androgens may promote pubertal growth. To assess this possibility, we administered dehydroepiandrosterone (DHEA) enanthate in monthly im injections in a dose of 70 mg/m2 for 1 yr to five boys with constitutional short stature (aged 11-13 4/12 yr) and one boy (aged 13 4/12 yr) with panhypopituitarism (coincidentally receiving T4 and human GH). All had bone age delay of at least 3 yr and subnormal levels of DHEA and DHEA sulfate (DHEA-S) for their chronological age. Pretreatment growth velocity ranged from 3-5 cm/yr. After DHEA enanthate injection, plasma DHEA levels were increased 10-fold after 8 days, 2.6-fold after 15 days, and 1.8-fold after 22 days. At the same times, plasma DHEA-S concentrations were 14-, 6-, and 4-fold increased, respectively. There was no rise in plasma testosterone and delta 4-androstenedione, which remained at prepubertal levels. During the year of therapy and for 1 yr after therapy, there was no significant change in growth velocity, and the rate of skeletal maturation assessed by x-ray was not affected. Three of the five boys with constitutional short stature entered puberty within 1 yr after discontinuation of therapy. These results demonstrate that this long-acting form of DHEA administered for 1 yr did not raise plasma testosterone above prepubertal levels and did not accelerate either growth or skeletal maturation. These findings do not support the possibility that DHEA plays a role in normal growth.

Hormonal changes in puberty III: Correlation of plasma dehydroepiandrosterone, testosterone, FSH, and LH with stages of puberty and bone age in normal boys and girls and in patients with Addison's disease or hypogonadism or with premature or late adrenarche.

In 104 normal boys, aged 7 to 14 years (bone ages 5 to 15 years), plasma dehydroepiandrosterone (DHEA) rose from 52.7 at 7 years, to 112.0 ng/100 ml at 10 years. A further rise occurred at 12 years (188 ng/100 ml). In relation to the bone age, DHEA increased from a mean plasma level of 31.1 at a bone age of 5 years to 77.1 ng/100 ml at one of 7 years. Further increases were observed with mean values of 163.2 at a bone age of 11 years, and of 221.2 at a bone age of 12 years, with a maximum of 333.4 ng/100 ml at bone ages of 14-15 years. The first significant increase of plasma testosterone (T) was noted at a bone age of 12 years (54.8 ng/100 ml). The major rise of T was preceded by the rise of plasma LH and was accompanied by the rise of plasma FSH. Plasma DHEA and T were also measured in 123 normal girls, ages 6 to 13 years (bone ages 5 to 15 years). DHEA rose significantly from a mean level of 44.7 at 6 years, to 80.9 ng/100 ml at 8 years, with further increases between 9 and 10 years and between 10 and 11 years. In relation to bone age, DHEA increased significantly from a mean plasma concentration of 30.9 at a bone age of 5 years, to that of 58.6 ng/100 ml at 7 years. Further increases were observed with values of 191.1 at a bone age of 10 years and 485.6 ng/100 ml at a bone age of 13 years. The first significant rise of testosterone (T) occurred at 10 years of both chronological and bone age. DHEA rose before the increase of gonadotropins. The major rise of T at a bone age of 10 years occurred concurrently with increases in plasma FSH and LH. Low levels of DHEA were observed in Addison's disease. In hypogonadotropin hypogonadism and in anorchia, DHEA levels were normal, suggesting that DHEA is produced primarily in the adrenal gland. In seven girls with early adrenarche, plasma concentrations of DHEA were in the upper range of normal values, whereas T levels were within the normal range. Conversely in girls with late adrenarche, plasms DHEA was lower than normal but T was within the normal limits. The elevation of DHEA prior to the first signs of puberty suggests that DHEA may play a role in the maturation of the hypothalamic-hypophysealgonadal axis. However, the mechanism that triggers the secretion of DHEA is not known.

Melatonin radioimmunoassay.

A radioimmunoassay for melatonin has been developed after the raising of anti-melatonin antibodies in rabbits. The radioimmunoassay is specific and the sensitivity range is greater than the tadpole bioassay.?Author

Hormonal changes during puberty: V. Transient pubertal gynecomastia: abnormal androgen-estrogen ratios.

The plasma profiles of 8 hormones were followed over the course of prepuberty and puberty in 30 adolescent males who developed gynecomastia and 24 who did not. Throughout puberty, ratios of delta 4-androstenedione to estrone (E1) and estradiol (E2) were significantly lower in the gynecomastia group than in the control group. Similarly, ratios of dehydroepiandrosterone-sulfate to E1 and E2 were significantly lower in the gynecomastia group. In contrast, ratios of plasma testosterone to E1 and E2 as well as plasma progesterone and PRL concentrations, were similar in both groups. Because of the adrenal origin of dehydroepiandrosterone and its sulfate, and of peripheral conversion of adrenal androgens to E1 and to E2, it appears that either decreased adrenal production of androgens and/or increased conversion of dehydroepiandrosterone-sulfate and delta 4-androstenedione to estrogens cause transient gynecomastia in adolescent boys.

Differences between circadian and ultradian organization of cortisol and melatonin rhythms during activity and rest.

We compared the cortisol and melatonin circadian and ultradian rhythms in normal men using two approaches: 1) the men were exposed successively to two conditions, one normal and a second chosen to alter differently each of the hormones, i.e. complete bedrest for 34 h (supine, fasting, and under dim light), and 2) analyses of the rhythms using a combination of curve smoothing for the description of the 24-h rhythm, and peak detection and spectral analysis for the measurement of periodic phenomena. Blood was sampled every 30 min from 0700-0700 h. A diurnal rhythm was detected for both hormones, with different underlying frequencies. Plasma cortisol had an ultradian rhythm of 8 h. From 0000-0800 h (night) and 0830-1600 h (early day), the pulsatile activity and baseline values of cortisol were high, while from 1630-2400 h (late day), these variables were low. During complete bedrest, pulsatile activity and baseline values were even higher during the night period, and the nocturnal peak of cortisol, usually present between 0300-1000 h, was split in two, with an early peak at 0000-0400 h. There were two specific events during the day associated with synchronous, high amplitude pulses: awakening and eating at noon. No such pulses occurred at suppertime or when the men fasted. Melatonin secretion was organized around a 5.5-h period. In the rest condition, plasma melatonin values were higher during the night. The 24-h rhythms of cortisol and melatonin were temporally related. Plasma melatonin began to rise when plasma cortisol was at its lowest, it peaked when cortisol began to rise, and it began to decrease when cortisol reached its peak, with a 5-h phase delay between plasma cortisol and melatonin rise at night. In summary, melatonin and cortisol rhythms have different ultradian frequencies, suggesting an intrinsic difference in the mechanisms controlling their secretion. In addition, their responses to restricted physical activity in an environment with dim light were completely different; for plasma melatonin, the change was primarily quantitative, with an increase in total production especially at night, while for plasma cortisol, there was more of a qualitative change, with different patterns of pulsatile activity and possible splitting of the nocturnal peak. The differences in the ultradian organization of these two hormones imply that the correlation between their peaks must depend on a third factor, which is likely to be the 24-h organization of the day.

Longitudinal monitoring of bone mass accumulation in healthy adolescents: evidence for a marked reduction after 16 years of age at the levels of lumbar spine and femoral neck in female subjects.

The amount of skeletal mass acquired during adolescence is one of the most important determinants for the risk of postmenopausal and involutional osteoporosis. In both sexes, a large variance in bone mineral density (BMD) and content (BMC) is observed among healthy individuals at the beginning of the third decade. To determine the crucial pubertal years during which bone mass accumulation mainly occurs, we longitudinally monitored the gain in BMD/BMC at clinically important sites, such as lumbar spine and femoral neck, with respect to osteoporotic fracture risk. The changes in BMD (grams per cm2) and BMC (grams) were determined at 1-yr intervals at the level of lumbar spine vertebrae (L2-L4), femoral neck, and midfemoral shaft, using dual energy x-ray absorptiometry (Hologic QDR 1000), in 198 healthy adolescents (98 females and 100 males), aged 9-19 yr. Mean daily energy and calcium intakes, height, weight, and body mass index of the studied cohort were within the normal range for age. In females, the increment rate in BMD/BMC was particularly pronounced over a 3-yr period, i.e. from 11-14 yr of age. This increment dramatically fell after 16 yr and/or 2 yr after menarche. The mean gains in lumbar, femoral neck, and midfemoral shaft BMD were not statistically significant between 17-20 yr. In males, the gain in BMD/BMC was particularly high over a 4-yr period, i.e. from 13-17 yr. Then the increment rate markedly declined, but remained significant between 17-20 yr for L2-L4 BMD/BMC and midfemoral shaft BMD. In contrast, no significant increase was observed for femoral neck BMD. An impressive interindividual variation was observed between the yearly height increment and the bone mass accumulation. The bone mass-height gains relationship during puberty evolved according to a loop pattern, with maximal variance at Tanner stages P3-P4. This longitudinal study delineates the crucial pubertal years during which the skeletal mass accumulates at high, but various, rates at skeletal sites where the consequences of the osteoporosis are particularly dramatic. Furthermore, the results indicate that in a cohort of healthy females with apparently adequate intakes of energy and calcium, bone mass accumulation is drastically reduced by 16 yr of age in both lumbar spine and femoral neck.

Lack of bromocriptine-induced reduction of predicted height in tall adolescents.

Fifteen girls and five boys with excessive predicted adult height (chronological age, 10.1-14.6 yr; bone age, 11.0-14.0 yr) were treated with bromocriptine (two doses; 2.5 mg/day) to reduce their final height. After a mean treatment period of 1.14 yr (range, 0.6-1.75 yr) we did not find a reduction of predicted adult height [difference, -0.5 +/- 3.5 (+/- SD) cm according to Bayley and Pinneau's tables (P = NS) and +0.2 +/- 2.5 (+/- SD) cm according to the method of Tanner (P = NS)]. Mean peak plasma GH concentrations after TRH administration before and during bromocriptine were 51.5 +/- 49.4 and 58.5 +/- 50.7 mU/L, respectively. The wide range of the GH values may be explained by physiological variation in this age group. After ingestion of 2.5 mg bromocriptine a significant increase in plasma GH occurred within 3 h in six adolescents tested. Our results do not support the concept that bromocriptine may reduce predicted adult height in tall adolescents by decreased GH secretion or acceleration of skeletal maturation.

Radioimmunological determination of urinary melatonin in humans: correlation with plasma levels and typical 24-hour rhythmicity.

To study melatonin secretion by a gentle noninvasive method, a simple and quick RIA procedure to analyze melatonin in small urine volumes has been developed. Urinary extracts were prepurified by alkaline washes, and melatonin content was determined by RIA. The specificity of urinary melatonin determinations was confirmed by both thin layer chromatography and by gas chromatography-mass spectrometry. In the present study, we compared the amount of melatonin excreted in urine with plasma levels in 140 specimens from 13 adult volunteers. Comparisons showed a very good correlation between plasma levels at midnight and nocturnal excretion of urine, indicating the biological relevance of melatonin determination in urine. Our studies show further that urinary melatonin excretion displays the characteristic circadian rhythm usually observed in plasma. On the average, melatonin excretion is greatest between 2300--0300 h. The total 24-h excretion of melatonin varies considerably among different individuals.