Chronic administration of neuropeptide Y into the lateral ventricle of C57BL/6J male mice produces an obesity syndrome including hyperphagia, hyperleptinemia, insulin resistance, and hypogonadism.

Neuropeptide Y (NPY) is involved in the central regulation of appetite, sexual behavior, and reproductive function. We have previously shown that chronic infusion of NPY into the lateral ventricle of normal rats produced an obesity syndrome characterized by hyperphagia, hyperinsulinism and collapse of reproductive function. We further demonstrated that acute inhibition of LH secretion in castrated rats was preferentially mediated by the NPY receptor subtype 5 (Y(5)). In the present study, the effects of chronic, central infusion of NPY, or the mixed Y2-Y5 agonist PYY(3-36), were evaluated both in normal male C57BL/6J mice and Sprague-Dawley rats. After a 7-day infusion to male mice, both NPY and PYY(3-36) at 5 nmol per day, induced marked hyperphagia leading to significant increases in body and fat pad weights. Furthermore, both compounds markedly reduced several markers of the reproductive axis. In the rat study, PYY(3-36) was more active than NPY to inhibit the pituitary-testicular axis, confirming the importance of the Y5 subtype for such effects. In the mouse, chronic NPY infusion induced a sustained increase in corticosterone and insulin secretion. Plasma leptin levels were also markedly increased possibly explaining the observed reduction in gene expression for hypothalamic NPY. Gene expression for hypothalamic POMC was reduced in the NPY- or PYY(3-36)-infused mice, suggesting that NPY exacerbated food intake by both acting through its own receptor(s), and reducing the satiety signal driven by the POMC-derived alpha-MSH. The present study in the mouse suggests in analogy with available rat data, that constant exposure to elevated NPY in the hypothalamic area unabatedly enhances food intake leading to an obesity syndrome including increased adiposity, insulin resistance, hypercorticism, and hypogonadism, reminiscent of the phenotype of the ob/ob mouse, that displays elevated hypothalamic NPY secondary to lack of leptin negative feedback action.

Hyperoxia increases leptin production: a mechanism mediated through endogenous elevation of corticosterone.

Leptin, a cytokine involved in the regulation of food intake, has been reported to be decreased in lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis and increased in critically ill patients with sepsis. We investigated the role of leptin during hyperoxia in mice, which results in alveolar edema, severe weight loss, and death within 3-4 days. In oxygen-breathing mice, serum leptin was increased six- to sevenfold and its mRNA was upregulated in white adipose tissue. Leptin elevation could not be attributed to changes in circulating tumor necrosis factor-alpha but was completely dependent on endogenous corticosterone elevation because adrenalectomized mice did not exhibit any increase in leptin levels. Using leptin-deficient mice and wild-type mice treated with anti-leptin antibody, we demonstrate that weight loss was leptin independent. Lung damage was moderately attenuated in leptin-deficient mice but was not modified by anti-leptin antibody or leptin administration, suggesting that leptin does not play an essential role in the direct and short-term effects of oxygen-induced injury.

GnRH antagonists: a new generation of long acting analogues incorporating p-ureido-phenylalanines at positions 5 and 6.

A series of antagonists of gonadotropin-releasing hormone (GnRH) of the general formula Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph/4Amf(P)-D4Aph/D4Amf(Q)-Leu-ILys-Pro-DAla-NH2 was synthesized, characterized, and screened for duration of inhibition of luteinizing hormone release in a castrated male rat assay. Selected analogues were tested in a reporter gene assay (IC50 and pA2) and an in vitro histamine release assay. P and Q contain urea/carbamoyl functionalities designed to increase potential intra- and intermolecular hydrogen bonding opportunities for structural stabilization and peptide/receptor interactions, respectively. These substitutions resulted in analogues with increased hydrophilicity and a lesser propensity to form gels in aqueous solution than azaline B [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Atz)-D4Aph(Atz)-Leu-ILys-Pro-DAla-NH2 with Atz = 3'-amino-1H-1',2',4'-triazol-5'-yl, 5], and in some cases they resulted in a significant increase in duration of action after subcutaneous (s.c.) administration. Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(L-hydroorotyl)-D4Aph(carbamoyl)-Leu-ILys-Pro-DAla-NH2 (acetate salt is FE200486) (31) and eight other congeners (20, 35, 37, 39, 41, 45-47) were identified that exhibited significantly longer duration of action than acyline [Ac-D2Nal-D4Cpa-D3Pal-Ser-4Aph(Ac)-D4Aph(Ac)-Leu-ILys-Pro-DAla-NH2] (6) when administered subcutaneously in castrated male rats at a dose of 50 microg in 100 microL of phosphate buffer. No correlation was found between retention times on a C18 reverse phase column using a triethylammonium phosphate buffer at pH 7.0 (a measure of hydrophilicity) or affinity in an in vitro human GnRH report gene assay (pA2) and duration of action. FE200486 was selected for preclinical studies, and some of its properties were compared to those of other clinical candidates. In the intact rat, ganirelix, abarelix, azaline B, and FE200486 inhibited plasma testosterone for 1, 1, 14, and 57 days, respectively, at 2 mg/kg s.c. in 5% mannitol (injection volume = 20 microL). Based on the information that 31, 33, 35 and 37 were significantly shorter acting than acyline or azaline B after intravenous administration (100 microg/rat), we surmised that the very long duration of action of the related FE200486 (for example) was likely due to unique physicochemical properties such as solubility in aqueous milieu, comparatively low propensity to form gels, and ability to diffuse at high concentrations in a manner similar to that described for slow release formulations of peptides. Indeed, in rats injected s.c. with FE200486 (2 mg/kg), plasmatic concentrations of FE200486 remained above 5 ng/mL until day 41, and the time after which they dropped below 3 ng/mL and plasma LH levels started rising until full recovery was reached at day 84 with levels of FE200486 hovering around 1 ng/mL. Additionally, FE200486 was less potent at releasing histamine from isolated rat mast cells than any of the GnRH antagonists presently described in preclinical reports.

Stimulation of the gonadotropic axis by the neuropeptide Y receptor Y1 antagonist/Y4 agonist 1229U91 in the male rat.

Neuropeptide Y (NPY) is a highly potent orexigenic substance that is also known to modulate gonadotropin secretion. Five receptor subtypes for NPY have been identified, and a potent antagonist for the receptor subtype 1 (Y1), 1229U91, also known as GW1229 or GR231118, has been described. Subsequently, 1229U91 was also shown to represent a highly potent agonist for the Y4 receptor subtype. Very unexpectedly, intracerebroventricular administration of 1229U91 elicited an intense, dose-dependent surge of both luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in intact male rats that lasted for 6 h. Such stimulation was absent when a potent gonadotropin-releasing hormone antagonist was administered systemically, suggesting that 1229U91 acts centrally to stimulate gonadotropin-releasing hormone release. 1229U91 administration had no effect on growth hormone, thyroid-stimulating hormone, and corticosterone secretions. In addition to 1229U91, four other parent dimer molecules described earlier produced a marked and sustained stimulation of LH when injected intracerebroventricularly that was proportional to their binding affinity for the Y4 receptor. Central administration of the specific Y1 antagonist BIBO3304 (20 microgram) had no effect on LH secretion, making it unlikely for 1229U91 to stimulate LH secretion by an antagonistic action on the Y1 receptor subtype, thus suggesting a Y4 receptor mediation. In conclusion, the 1229U91 molecule displays an interesting conformational epitope that is able to generate large LH surges, possibly by activating Y4 or Y4-like receptor subtypes or by acting on a NPY receptor unrelated target.

Modulation of human cytotrophoblastic leptin secretion by interleukin-1alpha and 17beta-oestradiol and its effect on HCG secretion.

To investigate the role of leptin during pregnancy, we assessed leptin production by pure cultured human cytotrophoblastic cells (CTB), its regulation by cytokines and 17beta-oestradiol and its effects on human chorionic gonadotrophin (HCG) secretion. Purified CTB from first trimester placenta were incubated in duplicates in the presence or absence of cytokines or 17beta-oestradiol. Medium was harvested on day 2 and the culture stopped on day 4. Results were corrected for protein content of each individual well and expressed as percent of controls per day (mean +/- SEM). Basal CTB leptin production was 25.2 +/- 2.6 (ng/mg prot). In comparison with controls, leptin production was stimulated to 320 +/- 16% (P < 0.0001) and 195 +/- 3.2% (P < 0.0004) by 3 and 10 ng/ml of interleukin-1alpha respectively. 17beta-oestradiol 10(-6) to 10(-9) mol/l increased basal leptin production 5-9-fold, while 10(-5) mol/l had no such effect. Basal CTB HCG secretion was 5722 +/- 1055 (mIU/mg prot). There was a dose-dependent leptin-induced increase in HCG secretion (P = 0.0039) reaching a 5-fold increase with a leptin concentration of 1 microg/ml (P < 0.006). Gonadotrophin-releasing hormone (GnRH) 8.5 x 10(-8) mol/l significantly increased HCG secretion to 140 +/- 21% of controls (P = 0.031). Cetrorelix (0.1 microg/ml) inhibited leptin-induced HCG secretion (P = 0.0028).

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.

Recombinant forms of rat and human luteinizing hormone and follicle-stimulating hormone; comparison of functions in vitro and in vivo.

We have previously described the preparation, purification and partial characterization of recombinant (rec) forms of rat luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In the present study, the special functional features of these hormones were studied further, in vitro and in vivo, and compared with human recLH and recFSH, as well as with human urinary choriongonadotropin (hCG) and rat pituitary LH (NIDDK-RP3). In radioreceptor assay, the affinity of hCG binding to rat testis membranes was 5-fold higher than that of human recLH and 100-fold higher than that of rat recLH. In in vitro bioassay, using dispersed adult mouse interstitial cells or a mouse Leydig tumor cell line (BLT-1), hCG and human recLH were 10- to 20-fold more potent than rat recLH. Correspondingly, rat pituitary LH was about 10-fold less potent than rat recLH, and evoked a maximum testosterone response that was about half of that elicited by the other LH/CG preparations. Rat recFSH was about 10-fold less potent than human recFSH in stimulating cAMP production of a mouse Sertoli cell line (MSC-1) expressing the recombinant rat FSH receptor. The circulating half-times (T1/2) of rat and human rec hormones were assessed after i.v. injections into adult male rats rendered gonadotropin-deficient by treatment with a gonadotropin-releasing hormone antagonist. A novel immunometric assay was used for the rat FSH measurements. In the one-component model the T1/2 values of rat and human recLH were 18.2 +/- 1.9 min (n = 7) and 44.6 +/- 3.1 min (n = 7) respectively and those of rat and human recFSH were 88.4 +/- 10.7 min (n = 6) and 55.0 +/- 4.2 min (n = 6) respectively; the two-component models revealed similar differences between the rec hormone preparations. Collectively, rat recLH was eliminated significantly faster from the circulation than human recLH (P < 0.0001). In contrast, the elimination of rat recFSH was significantly slower than that of human recFSH (P = 0.02). In conclusion, rat recFSH and rat recLH display lower biopotencies per unit mass than the respective human hormones in vitro, and also in vivo for LH. This is paralleled by shorter T1/2 of rat recLH than the respective human hormone in the circulation, whereas human recFSH has a shorter T1/2 than human FSH. The special functional features of the rat rec gonadotropins emphasize the use of these preparations on studies of gonadotropin function in the rat, an important animal model for reproductive physiology.

Identification of high-potency neuropeptide Y analogues through systematic lactamization.

In the pursuit of potent analogues of neuropeptide Y (NPY) that are selective for the Y1 receptor subtype, two lactam bridge scans of a centrally truncated parent compound were synthesized. A single lactam bridge (gamma-carboxyl of Glu to epsilon-amino of Lys) extending from residues i to i + 3 or i to i + 4 of the proposed alpha-helical region (residues 25-31 of NPY) was introduced in des-AA7-24[Gly6]NPY. Cyclogues (contraction of cyclic analogues), which were approximately one-half the size of native NPY, were initially screened for binding affinity at two discrete NPY receptor types using human neuroblastoma cell membranes, SK-N-MC and SK-N-BE2. Exploitation of the subtle differences present on each receptor type allowed for the identification of cyclogues which bound specifically to Y1 receptors with increased affinity when compared to the corresponding linear parent analogue, while one short Y1 specific cyclogue, des-AA2,3,5,7-24cyclo-(26/29)[Gly6,Glu26,Lys2 9,Pro34]NPY, bound with Ki = 16 nM. Other cyclogues showed distinct preference for Y2 receptors and bound in the low-nanomolar range. Functionally, the compounds inhibited the norepinephrine-stimulated accumulation of cAMP indicating that all acted as agonists with varying potencies.

[Effect of gonadotrophins, testosterone and growth hormone on spermatogenesis in two hypophysectomy patients]. / Effet des gonadotrophines, de la testostérone et de l'hormone de croissance sur la spermatogenèse chez deux patients hypophysectomisés.

Human chorionic gonadotrophin (hCG), human menopausal gonadotrophins (hMG) and somatotrope hormone (STH) were used for three months to induce spermatogenesis in a patient with azoospermia following hypophysectomy for Cushing's syndrome. Azoospermia reappeared when testosterone was substituted for hCG, despite continued treatment with FSH and STH. In a second patient who had undergone hypophysectomy for a craniopharyngioma presenting residual oligospermia, STH alone was uneffective in improving the sperm count. In both patients, the level of IGF-1 in seminal fluid was unchanged by STH despite increased serum IGF-1. The use of STH to induce spermatogenesis is discussed in light of its capacity to increase testosterone synthesis is response to hCG.

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.

Cyclic AMP increases the release of parathyroid hormone-related protein from a lung-cancer cell line.

Parathyroid hormone-related protein (PTHrP) plays an important role in the pathogenesis of malignant hypercalcemia by stimulating bone resorption and/or renal tubular reabsorption of calcium. In cultured cancer cells, its production can be influenced by various factors or ions, but the regulation of its production is still poorly understood. We investigated the effects of stimulators of cAMP synthesis on PTHrP release by a human lung squamous-carcinoma cell line (BEN). In superfused cells grown on microcarrier beads, PTHrP production was significantly increased after incubation with calcitonin for only 20 min. The release of immunoreactive and bioactive PTHrP was increased by incubating the cells with forskolin, 3-isobutyl-1-methylxanthine or dibutyryl cAMP even in the presence of the protein-synthesis inhibitor cycloheximide for 6 hr. The calcitonin-mediated stimulation was not accompanied by concomitant changes in PTHrP mRNA. The microfilament-disrupter cytochalasin D was shown to enhance the basal and calcitonin-induced production of PTHrP. These results indicate that stimulators of cAMP synthesis enhanced PTHrP release by BEN cells.

Intracellular gonadotropin-releasing hormone immunoreactivity in gonadotrophs of intact or castrated male rats: semiquantitative estimation of testosterone and GnRH antagonist treatment.

Changes in intracellular gonadotropin-releasing hormone (GnRH) immunoreactivity (GnRH-IR) in pituitary gonadotrophs were assessed by the use of a semiquantitative immunocytochemical method in male rats undergoing various manipulations known to greatly modify gonadotropin secretion. In basal conditions, immunoreactive GnRH was localized in the cytoplasmic matrix, the secretory granules and the nucleus of these cells. Following intravenous stimulation with GnRH (100 ng i.v.), the GnRH-IR increased rapidly in all these three subcellular compartments, peaking at 15 min. In untreated, long-term castrated rats, GnRH-IR increased both in the basal state and after administration of GnRH. Injection of the castrated rats with testosterone propionate reduced the observed GnRH-IR to the level observed in intact rats. Acute or chronic treatment of castrated rats with a potent GnRH antagonist rendered GnRH-IR completely undetectable in all the three previously positive subcellular compartments of gonadotrophs, and GnRH-IR did not reappear after stimulation with GnRH. In sum, the fact that modifications of GnRH immunoreactivity are observed in rat gonadotrophs: (1) confirms the GnRH internalization process; (2) suggests different sites of action for GnRH within the cell, and (3) demonstrates rapid clearance of intracellular GnRH after stimulation.

Reduction in testicular function in rats. I. Reduction by a specific gonadotropin-releasing hormone antagonist in fetal rats.

A gonadotropin-releasing hormone (GnRH) antagonist, when injected 24 h before sacrifice to rat fetuses, did not modify plasma testosterone concentrations in males on day 18 of gestation but it did on days 19, 20 and 21. This GnRH antagonist reduced plasma luteinizing hormone (LH) levels and increased pituitary LH content in both male and female 19-day-old fetuses from mothers adrenalectomized on day 14 of gestation. An inverse relationship between plasma testosterone and LH levels was noted in males and females, on days 19 and 21. These data suggest that the hypothalamic control of gonadotropic function is operating by day 19 of fetal life and that a negative feedback of testosterone on LH and probably GnRH release is also operating in rat fetuses on days 19 and 21 of gestation.

[Mechanism of action of gonadoliberin in pituitary gonadotropic cells]. / Mécanismes d'action de la gonadolibérine au niveau des cellules gonadotropes hypophysaires.

The gonadotropin-releasing hormone (GnRH) controls LH and FSH secretion by membrane receptor interaction followed by a transmission mechanism involving Ca2+ secretion and phosphoinositides hydrolysis. In the first step, GnRH binds to its receptor and induces the formation of aggregates of a certain number of hormone-receptor complexes. Some of these GnRH-receptor complexes are internalized. Then, the receptors are either degraded in lysosomes or recycled through inclusion into secretory granules in the area of the Golgi. Note that receptor internalization is not necessary for the LH response to GnRH that intervenes immediately after GnRH binding to its receptor. Binding of GnRH to membrane receptors provokes extracellular calcium flux into the cell. At the same time, an increase in intracellular Ca2+ from non-mitochondrial stock is observed. Intracellular Ca2+ is important for the initiation of LH response and then extracellular Ca2+ is necessary for a sustained response. GnRH also stimulates phosphoinositides hydrolysis (probably mediated by a G-protein) inducing inositol 1,4,5-triphosphate liberation. This component would participate in Ca2+i increase necessary for the early LH response. Another hydrolysis product is diacylglycerol (DAG) that binds principally to protein kinase C (PKC). On the one hand, this complex can activate "masked" GnRH receptors and participate in this manner to an up-regulation. On the other hand, it seems that FSH beta mRNA expression depends on PKC. GnRH secretion rythm modulates alpha, LH beta and FSH beta subunits mRNA expression, and secretion of these hormones. PKC could be an important regulator of these functions. DAG can also induce LH secretion, this mechanism uses PKC activation and requires gonadotrophin neosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Short-term and long-term effects of melatonin on GnRH-stimulated gonadotropin secretion in pituitaries of sexually maturing rats.

Melatonin administration has been shown to delay sexual maturation in male rats, through an action which involves decreased binding of gonadotropin-releasing hormone (GnRH) in the pituitary and lower pituitary gonadotropin contents. It has been suggested that melatonin must act at a level higher than the pituitary to provoke these effects, but a direct action of melatonin on the pituitary has not been excluded. Using a cell culture system, the gonadotroph responsiveness to GnRH was studied. Pituitaries were obtained either from rats chronically treated with melatonin and showing delayed sexual maturation, or from control rats. In vitro luteinizing hormone and follicle-stimulating hormone response to GnRH was significantly lower when pituitaries were obtained from melatonin-treated rats. However, this diminished response was directly proportional to the amount of gonadotropin contents in cells, so that relative responsiveness, calculated as the amount of gonadotropins released in relation to the gonadotropin content was similar in cells from control and melatonin-treated rats. It is concluded that the effect of melatonin on the pituitary of male rats results from a decrease of gonadotroph growth or gonadotropin synthesis, as a consequence of a change located at the hypothalamic rather than at the pituitary level. This conclusion is further supported by results showing that melatonin added directly in culture medium prior to GnRH does not modify the pituitary responsiveness to GnRH.

Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat. A quantitative autoradiographic study after cryoultramicrotomy.

To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2-60 min) after intravenous injection of mono-radioiodinated 125I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp6, Pro9 (Net), DesGly10]-GnRH produced comparable results. In contrast, the injection of 125I-[D-pGlu1, D-Phe2, Trp3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.

[Differentiation of pituitary cells in culture]. / Différenciation des cellules antéhypophysaires en culture.

Gonadotrophs were first detected at 18 days of gestation in normal rat fetus. Encephalectomy performed at 16 days of gestation did not modify the normal aspect of cells at term. In adenohypophysial primordia explanted from 13 days of gestation differentiated gonadotrophs were detected after culture (8 days) in medium containing insulin (minimal dose required: 0.5 microgram/ml) and transferrin (5 micrograms/ml). In contrast, in primordia explanted at 11 and 12 days of gestation, GnRH 10(-9) to 10(-12) M was required for the first 24 hours of culture to induce differentiation of cells which was obtained in synergy with insulin and transferrin. On the other hand, fetal hypothalamic GnRH and pituitary GnRH receptors were observed from 12 days of gestation which can explain the observations made on primordia explanted at 13 days. Lactotrophs first appeared at term in normal rat fetus. In vitro, differentiation of lactotrophs was not observed in primordia explanted from 13 days in the presence of insulin and transferrin alone, but it was induced by GnRH (10(-9) M) for the 24 hours of culture in the same medium. The action of GnRH was mediated through glycoproteins and specifically isolated alpha subunit. Indeed, purified LH alpha-subunit added in medium instead of GnRH induced differentiation of lactotrophs from 10(-9) M with an increase in the number of cells related to the dose of hormone. Differentiation of the two cell types is very linked in these culture conditions. Gonadotroph differentiation is regulated by an hypothalamic endocrine secretion whereas lactotroph differentiation is more dependent on a paracrine secretion.

Structure-function studies on human growth hormone. Evidence that tertiary structure is essential for biological activity.

The relationship between the conformation of human pituitary growth hormone (hGH), biological activity, and ligand binding activity was studied by comparing conformational details previously published on in vivo and in vitro studies of identical samples of hGH and its known derivatives. In vivo assays included the rat tibia test for somatotropic activity and the pigeon crop-sac assay for lactogenic hormone activity. Relative binding affinities were compared in radioimmunoassays using 125I-hGH as tracer with 1) anti-human chorionic somatomammotropin (hCS) serum (low discriminatory hybrid assay), 2) anti-hGH sera (in conventional assays), 3) monospecific anti-hGH serum (absence of cross-reaction with hCS) and 4) human anti-hGH sera obtained from GH-deficient patients on replacement therapy. In addition, binding affinities were examined in two receptor-binding assays, one specific for somatotropic activity (rabbit liver membranes, 125I-hGH), and the other, for lactogenic hormones (rabbit mammary membranes, 125I-oPRL). The conformational properties of native hGH and various chemically and enzymatically modified derivatives of the hormone were evaluated primarily from circular dichroism spectra, while conformational stabilities were estimated from the relative rates of tryptic digestion. Unfragmented, but chemically modified derivatives, exhibited good parallelism between retention or loss of native conformation and the in vivo potencies and in vitro binding affinities. None of the fragments of hGH showed activity in any of the radioreceptor assays or radioimmunoassays. Two derivatives of hGH, which contain gaps of 6 or 12 residues in the polypeptide backbone produced by partial enzymatic digestion, had full or increased in vivo potencies, full activities in the radioimmunoassays, and were the most active derivatives in both radioreceptor assays. One of these, missing the hexapeptide corresponding to residues 135-140, was also found to retain nearly all the conformational properties of native hGH. These studies proved further evidence that 1) retention by modified forms of hGH of a high degree of in vivo biological potency or in vitro binding affinity is causally related to the retention of most of the conformation and conformational stability of the molecule, and 2) the biologically active, receptor-binding and immunoreactive sites on the hGH molecule are 3-dimensional in nature.

Puberty in the rat: modulation by melatonin and light.

Although a role has been found for melatonin in species which have a seasonal reproductive cycle, very little is known on the role of melatonin in species such as the rat where seasonal cycles are a minor component of reproduction. But the rat is a photosensitive species, because it responds to changes in the lighting environment. Females do not become quiescent but they can have irregular estrous cycles, another way of controlling population dynamics. In this species, exogenous melatonin can exert an antigonadotropic action, providing conditions which apply to other species are also respected for the rat: melatonin must be given at the right time of the day, 9 to 12 hours after the onset of light, and at a given period of life, before the onset of puberty. Melatonin probably acts on the pattern of GnRH pulsatile secretion and the subsequent alterations of the hypothalamic-gonadal axis differ according to the sex of the animal. Endogenous melatonin rhythms are modified by the lighting environment, and results obtained with exogenous melatonin suggest that they could be one of the factors controlling timing of sexual maturation.