Induction of pituitary lactotrope differentiation by luteinizing hormone alpha subunit.

Addition of gonadotropin releasing hormone to cultures of fetal rat pituitary induced differentiation of lactotropes as revealed by immunocytochemistry. Antiserum to luteinizing hormone (LH) (recognizing native LH), but not antiserum to LH-beta (recognizing both native LH and its beta subunit), inhibited this induction. Further addition of highly purified LH-alpha subunit in culture medium also induced lactotrope differentiation. Thus, the alpha subunit may have a specific biological activity of its own with probable practical use in clinical investigations.

Differentiation of fetal rat somatotropes in vitro: effects of cortisol, 3,5,3'-triiodothyronine, and glucagon, a light microscopic and radioimmunological study.

Cortisol stimulates somatotrope differentiation in vitro. T3 and/or glucagon may also be involved. Fetal rat pituitary primordia were explanted at 14 days gestation and cultured for 7 days in medium supplemented with cortisol (50-500 nM), and either T3 (0.67 nM) or glucagon (0.5 nM). Also, to determine the time of first appearance of the somatotropes, explants were cultured 4, 5, or 6 days with cortisol alone. Immunoreactive somatotropes were detected by immunohistochemistry, and their size and number were estimated for each medium. GH was measured by RIA in explants and media. Immunoreactive somatotropes first appear at 18-19 days gestation. Their size and number depend on cortisol concentration: no cells at 50 nM, a few small ones at 100 nM, and many large ones at 250-500 nM. This progression was reflected by RIA of GH in explants and media, although small quantities were detected with 50 nM. The effect of T3 was only visible with a low dose of cortisol. With 100 nM cortisol, it increased the size and number of cells. Differentiation was also triggered with 50 nM cortisol plus T3. RIA detected significantly higher GH content and secretion after T3 stimulation. The decreases in number, size, and GH secretion and content elicited by glucagon were not significant, probably due to the high variability. Both techniques used provide similar information on somatotrope differentiation: stimulation by cortisol alone, or alternatively by a synergistic action between cortisol and T3.

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.

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.

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.

Chronic blockade of the melanocortin 4 receptor subtype leads to obesity independently of neuropeptide Y action, with no adverse effects on the gonadotropic and somatotropic axes.

Neuropeptide Y (NPY) is a powerful orexigenic factor, and alphaMSH is a melanocortin (MC) peptide that induces satiety by activating the MC4 receptor subtype. Genetic models with disruption of MC4 receptor signaling are associated with obesity. In the present study, a 7-day intracerebroventricular infusion to male rats of either the MC receptor antagonist SHU9119 or porcine NPY (10 nmol/day) was shown to strongly stimulate food and water intake and to markedly increase fat pad mass. Very high plasma leptin levels were found in NPY-treated rats (27.1 +/- 1.8 ng/ml compared with 9.9 +/- 0.9 ng/ml in SHU9119-treated animals and 2.1 +/- 0.2 ng/ml in controls). As expected, NPY infusion induced hypogonadism, characterized by an impressive decrease in seminal vesicle and prostate weights. No such effects were seen with the SHU9119 infusion. Similarly, whereas the somatotropic axis of NPY-treated rats was fully inhibited, this axis was normally activated in the obese SHU9119-treated rats. Chronic infusion of SHU9119 strikingly reduced hypothalamic gene expression for NPY (65.2 +/- 3.6% of controls), whereas gene expression for POMC was increased (170 +/- 19%). NPY infusion decreased hypothalamic gene expression for both POMC and NPY (70 +/- 9% and 75.4 +/- 9.5%, respectively). In summary, blockade of the MC4 receptor subtype by SHU9119 was able to generate an obesity syndrome with no apparent side-effects on the reproductive and somatotropic axes. In this situation, it is unlikely that hyperphagia was driven by increased NPY release, because hypothalamic NPY gene expression was markedly reduced, suggesting that hyperphagia mainly resulted from loss of the satiety signal driven by MC peptides. NPY infusion produced hypogonadism and hyposomatotropism in the face of markedly elevated plasma leptin levels and an important reduction in hypothalamic POMC synthesis. In this situation NPY probably acted both by exacerbating food intake through Y receptors and by reducing the satiety signal driven by MC peptides.

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)

Chronic administration of neuropeptide Y into the lateral ventricle inhibits both the pituitary-testicular axis and growth hormone and insulin-like growth factor I secretion in intact adult male rats.

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 in satiated animals, diet restriction or other unfavorable metabolic situations, such as diabetes, produce enhanced NPY gene expression and NPY release in the hypothalamus. Numerous studies have indicated that acute central administration of NPY results in various actions on LH secretion in the rat, either stimulatory or inhibitory. We recently demonstrated that chronic infusion of NPY into the lateral ventricle of adult intact female rats profoundly inhibited both the gonadotropic and somatotropic axes, with disruption of estrous cyclicity. Furthermore, we showed that central chronic infusion of NPY delayed sexual maturation in female rats. To analyze the effects of the same type of chronic NPY treatment on the pituitary-testicular axis, 45-day-old Sprague-Dawley male rats were implanted with stainless steel cannulas in the right lateral ventricle. Ten days later, Alzet osmotic minipumps were filled with different NPY solutions, adjusted to deliver 6, 18, or 36 micrograms/day, connected to the intracerebroventricular (icv) cannula, and sc implanted dorsally. The effects of these treatments were evaluated over 7 days. In one case, rats were castrated 5 days after initiation of NPY treatment, and the effect of castration was evaluated 2 days later. Chronic icv infusion of NPY produced the expected dose-related increases in food intake from 33.0 +/- 0.9 (basal) to 53.4 +/- 3.3 g/day (18 micrograms NPY/day) and body weight gain (5.7 +/- 0.7 to 10.5 +/- 1.2 d/day). As in female rats, this orexigenic action of NPY resulted in a significant dose-related decrease in pituitary weight, from 12.4 +/- 0.7 to 9.9 +/- 0.4 mg. The 7-day NPY infusion produced highly significant decreases in seminal vesicle weight (853 +/- 77 to 230 +/- 31 mg) and testis weight (3.82 +/- 0.09 to 3.18 +/- 0.15 g; P = 0.003). Plasma levels of testosterone (231 +/- 71 to 48 +/- 13 ng/dl), LH (20.7 +/- 3.7 to 9.1 +/- 1.2 ng/ml), and FSH (282 +/- 17 to 190 +/- 18 ng/ml) were markedly decreased at the 18 micrograms/day dosage, as also demonstrated for the 36 micrograms/day dosage. None of these effects was observed if vehicle was infused into the lateral ventricle instead of the NPY solution. When bilateral orchidectomy was performed 5 days after initiation of the NPY infusion (18 micrograms/day), the immediate LH and FSH rises usually seen after castration were seriously blunted.(ABSTRACT TRUNCATED AT 400 WORDS)

Chorionic gonadotropin, chorionic somatomammotropin, and prolactin in the uterine vein and peripheral plasma of pregnant rhesus monkeys.

Seven adult female rhesus monkeys were laparotomized at days 22, 42, and 157 of pregnancy and blood was collected from the uterine vein and peripheral circulation. Plasma samples were analyzed for monkey chorionic gonadotropin (mCG), monkey chorionic somatomammotropin (mCS), and prolactin by radioimmunoassay. Levels of mCG at day 22 of pregnancy were approximately 250 ng/ml; however, during the later stages of gestation mCG was either nondetectable or less than 0.7 ng/ml. There was no statistical difference in prolactin concentrations between days 22 and 42 of pregnancy, mean levels being between 176-424 ng/ml, but by day 157 prolactin levels of greater than 2,000 ng/ml were recorded. No statistical difference existed between peripheral and uterine vein concentrations of either mCG or prolactin at any of the stages of gestation examined. At day 22, mCS was not detectable; however, at day 42 of gestation mCS titers averaged 1.5 micrograms/ml and 2.3 micrograms/ml in the peripheral and uterine vein plasma, respectively. A statistically significant mCS increase occurred by day 157, levels in the periphery and uterine vein averaging 11.0 micrograms/ml and 16.3 micrograms/ml, respectively. Uterine vein titers of mCS were significantly higher than peripheral titers at both days 42 and 157. Thus, the highest levels of mCG were present during early pregnancy, whereas the highest levels of mCS and prolactin were present during late pregnancy.

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.

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.

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.

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.

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.

Evidence that the inhibition of luteinizing hormone secretion exerted by central administration of neuropeptide Y (NPY) in the rat is predominantly mediated by the NPY-Y5 receptor subtype.

A number of studies have indicated that neuropeptide Y (NPY) is a central regulator of the gonadotropic axis, and the Y1 receptor was initially suggested to be implicated. As at least five different NPY receptor subtypes have now been characterized, the aim of the present study was to reinvestigate the pharmacological profile of the receptor(s) mediating the inhibitory action of NPY on LH secretion by using a panel of NPY analogs with different selectivity toward the five NPY receptor subtypes. When given intracerebroventricularly (icv) to castrated rats, a bolus injection of native NPY (0.7-2.3 nmol) dose-dependently decreased plasma LH. Peptide YY (PYY; 2.3 nmol) was as potent as NPY, suggesting that the Y3 receptor is not implicated. Confirming previous data, the mixed Y1, Y4, and Y5 agonist [Leu31,Pro34]NPY (0.7-2.3 nmol) inhibited LH release with potency and efficacy equal to those of NPY. Neither the selective Y2 agonist C2-NPY (2.3 nmol) nor the selective Y4 agonist rat pancreatic polypeptide affected plasma LH, excluding Y2 and Y4 subtypes for the action of NPY on LH secretion. The mixed Y4-Y5 agonist human pancreatic polypeptide (0.7-7 nmol) as well as the mixed Y2-Y5 agonist PYY3-36 (0.7-7 nmol) that displayed very low affinity for the Y1 receptor, thus practically representing selective Y5 agonists in this system, decreased plasma LH with potency and efficacy similar to those of NPY, indicating that the Y5 receptor is mainly involved in this inhibitory action of NPY on LH secretion. [D-Trp32]NPY, a selective, but weak, Y5 agonist, also inhibited plasma LH at a dose of 7 nmol. Furthermore, the inhibitory action of NPY (0.7 nmol) on LH secretion could be fully prevented, in a dose-dependent manner (6-100 microg, icv), by a nonpeptidic Y5 receptor antagonist. This antagonist (60 microg, icv) also inhibited the stimulatory action of NPY (0.7 nmol) on food intake. The selectivity of PYY3-36, human PP, [D-Trp32]NPY, and the Y5 antagonist for the Y5 receptor subtype was further confirmed by their ability to inhibit the specific [125I][Leu31,Pro34]PYY binding to rat brain membrane homogenates in the presence of the Y1 receptor antagonist BIBP3226, a binding assay system that was described as being highly specific for Y5-like receptors. With the exception of [D-Trp32]NPY, all analogs able to inhibit LH secretion were also able to stimulate food intake. Taken together, these results indicate that the Y5 receptor is involved in the negative control by NPY of the gonadotropic axis.

alpha and beta glycoprotein hormone subunits (hLH, hFSH, hCG) in the serum and pituitary of the human fetus.

Utilizing specific radioimmunoassay and gel chromatographic separation methods, the concentration of a glycoprotein hormone subunit and the beta subunit of hCG, hLH, and hFSH was determined. The alpha glycoprotein hormone subunit is present throughout gestation in the serum and pituitary of the human fetus; little or no subunit is apparent in contrast to that of intact LH and FSH.

Pattern of plasma ACTH, hGH, and cortisol during menstrual cycle.

The pattern of secretion of plasma ACTH, hGH, TSH, LH, FSH and cortisol was studied in 12 menstrual cycles, representing 5 normal volunteers. Results were plotted by taking the LH-FSH midcycle peak as day 0. The typical menstrual cyclic LH and FSH pattern was observed in each case. ACTH, cortisol and hGH varied significantly throughout the menstrual cycle. ACTH was characterized by a decrease during the follicular phase, a nadir at day -2, followed by a significant increase to a peak at day 0, then a subsequent decrease and constant levels during the luteal phase until day +8. Cortisol was lowest in the follicular phase until day -4, and highest from day -2 to day 0. During the luteal phase, cortisol remained constant but was significantly higher than in the follicular phase (days -7 to -4). hGH showed a significant increase during the periovulatory period (days -3 to +3). No significant changes of plasma TSH were observed. These results suggest that pituitary hormones other than gonadotropins may be involved in the ovulatory mechanism, and reveal a degree of stimulation of the pituitary-adrenal axis without establishing whether the effect is direct or indirect.

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.