LH Secretion In Rats Research Articles

How does apelin affect LH levels? An investigation at the level of GnRH and KNDy neurons

Hypothalamic control of reproduction relies on GnRH and kisspeptin (KP) secretions. KP neurons are sensitive to sex steroids and metabolic status and their distribution overlaps with neurons producing apelin, a metabolic hormone known to decrease LH secretion in rats. Here, we observed neuroanatomical contacts between apelin fibers and both KP and GnRH neurons in the hypothalamus of male rodents. Intracerebroventricular apelin infusion for 2 weeks in male mice did not decrease LH levels nor did it affect gene expression for KP, neurokinin B and dynorphin. Finally, increasing apelin concentrations did not modulate Ca2+ levels of cultured GnRH neurons, while 10 μM apelin infusion on forskolin pretreated GnRH neurons revoked a rhythmic activity in 18% of GnRH neurons. These results suggest that acute apelin effect on LH secretion does not involve modulation of gene expression in KP neurons but may affect the secretory activity of GnRH neurons.

Neuroendocrine control of the pituitary gonadotropic function in experimental diabetes

The hypothalamo-pituitary-gonadal system was examined in male and female rats with experimental diabetes in­duced by streptozotocin (STZ). Injection of STZ caused a decrease of testosterone (T) concentration and of T nuclear receptors in the pituitary. The levels of luteinizing and follicle stimulating hor­mones (LH and FSH) in the blood of diabetic rats did not differ from those in intact animals. In vitro experiments showed that the development of diabetes did not change the basal secretion of LH by the pituitary in males. Maximal response to LH-RH was record­ed in control males after 3-hour incubation, whereas the rate of LH secretion in experimental rats did not differ from basal values. In­jection of STZ to cycling females disordered the estrous cycle and involved decreases of the basal and cyclic secretion of LH, FSH, and sex hormones. The concentrations of estradiol nuclear receptors in the preoptic anterohypothalamic region and pituitary decreased, whereas the number of T-binding sites decreased only in the pitui­tary. Sex hormone-stimulated gonadotropin wave in oophorect- omized females was decreased in diabetes, which was due to changed activity of the LH-RH producing system. The authors hy­pothesize that changes in the mechanism of regulation of the hy­pothalamo-pituitary-gonadal system in experimental diabetes are re­lated to pituitary disorders in males, whereas changed basal and cy­clic secretion of LH and FSH in females is caused by disordered activity of the LH-RH production and receptor binding at the level of the hypothalamo-pituitary complex.

OR16-5 Feedback on LH in Testosterone-Clamped Men Depends on the Mode of Testosterone Administration and Body Composition

Context. Investigations of testosterone (T)-mediated feedback on LH secretion are few and contradictory. Discrepancies may reflect the choice of androgen, as well as dose, route and/or pattern (continuous vspulsatile) of administration. Moreover, for controlled T feedback studies, endogenous T secretion must be shut down and replaced by iv T infusion. Objective. The goal of this study was to quantify feedback in relation to the infused T pattern (pulsatile vs nonpulsatile) with the hypotheses that: 1) feedback by T pulses is less inhibitory, 2) T feedback operates partly at the pituitary level, 3) T feedback is controlled in part by body composition. Design. This was a placebo-controlled, blinded, and prospectively randomized crossover study comprising 16 men (age range 23-54 yr and BMI between 22.3-34.2 kg/m2). Subjects received ketoconazole (1600 mg over 22 h to block endogenous T secretion: serum T below 85 ng/dL) followed by pulsatile or continuous iv T addback (total T dose 5 mg/22 h). Cortisol inhibition was treated with low-dose dexamethasone. Setting. The study was performed in a Clinical Translational Research Unit. Interventions. Subjects underwent 14 h of blood sampling at 10-min intervals, starting at 0600 h. T was given continuously starting at 0900 h or as 45-min squarewave pulses every 90 min. To test pituitary feedback, a bolus iv injection of 33 ng/kg GnRH was given at 1630 h. Outcome measures. Log-transformed LH and T concentration ratios before and after GnRH administration. In addition, a four-parameter logistic regression between serum T concentration and LH secretion rate (calculated by deconvolution analysis) was used to independently estimate feedback of T on LH. Results. Despite higher T (total, bioavailable and free T) concentrations during pulsatile feedback, LH concentrations and secretion rates, whether driven by endogenous or exogenous GnRH, were similar to those during continuous infusion, indicating diminished pulsatile T feedback. This interpretation was corroborated by the right-shift (i.e. towards higher T concentrations) of the T-LH inhibitory dose-response curve. During continuous and pulsatile T infusion, LH concentrations and LH:T ratios depended positively on BMI to a similar extent before and after GnRH administration. Under both feedback regimens, basal but not pulsatile LH secretion correlated negatively with CT-estimated abdominal visceral fat mass. Age was not a significant covariate over the limited range explored. Conclusion. In summary, pulsatile T is less effective as a negative feedback signal on LH secretion than continuous T administration, as demonstrated by two independent methods. This difference in feedback strength was evident for exogenous GnRH-stimulated LH release as well, and thus occurs probably in part at the pituitary level, while not excluding additional hypothalamic involvement.

Naloxegol, an opioid antagonist with reduced CNS penetration: Mode-of-action and human relevance for rat testicular tumours

Naloxegol is an opioid antagonist which has been developed for the treatment of patients with opioid induced constipation. In the nonclinical safety program naloxegol was shown to have a very benign toxicity profile. In the rat, but not the mouse, 2-year carcinogenicity study a change in tumour pattern with an increase in testicular Leydig cell tumours (LCT) was observed after dosing at high (supra-pharmacological) concentrations. To establish the basis of the increase in LCT and to assess its potential relevance to humans, studies to exclude and potentially identify mode-of-action (MoA) were performed. A genotoxic mechanism was ruled out following negative results in the Ames, mouse lymphoma, and micronucleus assays. An effect on androgen metabolism was excluded since the treatment of rats with naloxegol for 14days did not result in any induction of CYP protein levels. It was demonstrated that administration of centrally restricted opioid antagonists naloxegol or methylnaltrexone at high doses induced an increase in LH release with no clear increase in testosterone, in contrast to the centrally acting opioid antagonist naloxone, which showed marked increases in both LH and testosterone. LCT due to increased LH stimulation is common in rats but not documented in humans. Collectively, the lack of genotoxicity signal, the lack of androgen effect, the increase in LH secretion in rats, which is no considered to be relevant for LCT formation in humans, and high margins to clinical exposures, the observed increase in LCT in the rat is not expected to be clinically relevant.

Development of a Methodology for and Assessment of Pulsatile Luteinizing Hormone Secretion in Juvenile and Adult Male Mice

Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-μL fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events (number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.

Effect of royal jelly in ovo injection on embryonic growth, hatchability, and gonadotropin levels of pullet breeder chicks

The objectives of this study were to compare the following: (1) hatchability, (2) chicks' body and internal organs weights, and (3) plasma gonadotropin levels of hatchlings after in ovo administration of royal jelly (RJ) on Day 7 of incubation. Fertile eggs (n = 270) were injected into the air sac or yolk sac with 0.5 mL normal saline solution consisting of four formulations (normal saline solution with antibiotics, ultrafiltrate RJ, pure RJ, and RJ with antibiotics). The eggs were randomly divided into nine groups of 30 eggs each: (i) C: the control eggs received no injection, (ii) ASA: air sac–injected eggs received normal saline solution with antibiotics, (iii) ARJ: air sac–injected eggs received pure RJ, (iv) ARJA: air sac–injected eggs received RJ with antibiotics, (v) ARJF: air sac–injected eggs received RJ ultrafiltrate solution, (vi) YSA: yolk sac–injected eggs received normal saline solution with antibiotics, and (vii) YRJ: yolk sac–injected eggs received pure RJ, (viii) YRJA: yolk sac–injected eggs received RJ with antibiotics, and (ix) YRJF: yolk sac–injected eggs received ultrafiltrate RJ solution. Hatchability rate was lower in ARJ (46.7%), ARJA (43.3%), ARJF (43.3%), and YRJF (46.7%) groups than in the control (80.0%; P < 0.05). Hatchability rate in ASA (70.0%), YSA (66.7%), YRJ (66.7%), and YRJA (63.3%) groups were comparable to the control (P > 0.05). In ovo injection of RJ and or RJ with antibiotics in both sacs increased chicks’ body weight (CWT), heart weight (HWT), and liver weight (LWT) and FSH and LH levels compared with control (P < 0.05). CWT in YRJ (37.02 g), YRJA (37.03 g), ARJ (36.82 g), and ARJA (36.89 g) groups were higher than control (34.9 g; P < 0.05). Similarly, HWT significantly increased in YRJ (0.22 g), YRJA (0.21 g), ARJ (0.20 g), and ARJA (0.20 g) in comparison to control (0.18 g; P < 0.05). In addition, LWT were higher in YRJ (0.83 g), YRJA (0.82g), ARJ (0.81g), and ARJA (0.81g) than control (0.72 g; P < 0.05). Six hours post-hatch, the mean plasma FSH and LH levels in ARJ (1.13 and 2.80 mIu/mL), YRJ (1.32 and 3.36 mIu/mL), ARJA (1.23 and 2.95 mIu/mL), and YRJA (1.31 and 3.28 mIu/mL) groups were higher than in the control (0.56 and 1.48 mIu/mL, P < 0.05). We concluded that in ovo administration of RJ or RJ with antibiotics might be an effective method to increase CWT, chicks' internal organs weights, and LH and FSH secretion rate without deleterious effect on hatchability. However, further research should be conducted to determine the putative endocrine disruptive effects of RJ and its byproducts.

Estradiol and LH secretion and ovulation rate in sows after PG600 treatment during weaning

Estradiol and LH secretion and ovulation rate in sows after PG600 treatment during weaning

Targeted pituitary overexpression of pituitary adenylate-cyclase activating polypeptide alters postnatal sexual maturation in male mice.

The neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) is present in high concentrations within the hypothalamus, suggesting that it may be a hypophysiotropic factor, whereas pituitary expression suggests a paracrine function. PACAP stimulates gonadotropin secretion and enhances GnRH responsiveness. PACAP increases gonadotropin α-subunit (αGSU), lengthens LHβ, but reduces FSHβ mRNA levels in adult pituitary cell cultures in part by increasing follistatin. PACAP stimulates LH secretion in rats; however, acceptance of PACAP as a regulator of reproduction has been limited by a paucity of in vivo studies. We created a transgenic mouse model of pituitary PACAP overexpression using the αGSU subunit promoter. Real-time PCR was used to evaluate PACAP, follistatin, GnRH receptor, and the gonadotropin subunit mRNA in male transgenic and wild-type mice of various ages. Transgenic mice had greater than 1000-fold higher levels of pituitary PACAP mRNA; and immunocytochemistry, Western blot, and ELISA analyses confirmed high peptide levels. FSH, LH, and testosterone levels were significantly suppressed, and the timing of puberty was substantially delayed in PACAP transgenic mice in which gonadotropin subunit and GnRH receptor mRNA levels were reduced and pituitary follistatin expression was increased. Microarray analyses revealed 1229 of 45102 probes were significantly (P < 0.01) different in pituitaries from PACAP transgenic mice, of which 83 genes were at least 2-fold different. Genes involved in small molecule biochemistry, cancer, and reproductive system diseases were the top associated networks. The GnRH signaling pathway was the top canonical pathway affected by pituitary PACAP excess. These experiments provide the first evidence that PACAP affects gonadotropin expression and sexual maturation in vivo.

Involvement of Neurokinin Receptors in the Control of Pulsatile Luteinizing Hormone Secretion in Rats

It has recently been shown that neurokinin B, a tachykinin, is associated with GnRH pulse generation in sheep and goats. The aim of the present study was to clarify the role of tachykinin receptors in the control of LH secretion in rats. To this end, we evaluated the effect of CS-003, an antagonist for all three neurokinin receptors (NK1, NK2 and NK3 receptors), on pulsatile LH secretion in both sexes of rats with different routes of administration. Both oral and third ventricular administration of CS-003 suppressed LH secretion in both sexes of gonadectomized animals. Furthermore, intact male rats with oral administration of CS-003 showed decreased serum testosterone levels, which might be due to suppressed LH secretion. None of the three subtype-specific neurokinin receptor antagonists showed a significant effect on LH secretion in ovariectomized rats when each antagonist was singly administered. The present results suggest that neurokinins play a role in the control of pulsatile GnRH/LH secretion via multiple neurokinin receptors in both male and female rats.

Neurokinin B Acts via the Neurokinin-3 Receptor in the Retrochiasmatic Area to Stimulate Luteinizing Hormone Secretion in Sheep

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Basal and pulsatile secretion of human luteinizing hormone--new methods for the analysis of endocrine secretion processes.

Serum concentrations of hormones show typical time-dependent patterns. The diurnal course of the serum concentrations of the luteinizing hormone (LH) was assessed in 35 healthy females monitoring individual concentrations every ten minutes for 24 hours during the follicular phase of their menstrual cycle. Based on these in vivo data, a mathematical procedure was developed for calculation of the momentary hormone secretion rate of an endocrine gland from the time course of the hormone serum concentrations. By means of this procedure, we were able to define the complete 24 hours secretion pattern of LH, including the fact, that basal and pulsatile secretion could be separated from each other in an objective manner. It is possible to calculate the total amount of LH which is secreted per LH-pulse and the basal LH-secretion rate. Two methods were developed to define the time of the onset of an LH-pulse with a preselected probability. Describing the hormonal secretion pattern with a mathematical model provides the basis for therapeutic application of these methods to program implantable hormone-pumps for the therapy of glandular insufficiency.

Acute Effects of Testosterone Infusion on the Serum Luteinizing Hormone Profile in Eumenorrheic and Polycystic Ovary Syndrome Adolescents

Little is known about the neuroendocrine effects of androgens on the GnRH-LH unit in females. Our objective was to evaluate androgen negative feedback on the GnRH-LH axis in eumenorrheic and polycystic ovary syndrome (PCOS) adolescents. We conducted a prospective, longitudinal, randomized, double-blind study at a pediatric endocrinology clinical research center. Seven nonobese PCOS adolescents and seven matched controls (C) were studied in the early follicular phase of three consecutive menstrual cycles or in three consecutive months. Pulsatile LH release was determined during saline [baseline (B)] and constant testosterone (T) infusions: low dose (T-LD) 0.75 and high dose (T-HD) 2.5 mg/12 h iv. Blood samples were drawn every 20 min overnight. LH (immunofluorometric assay) and T (electrochemiluminescence immunoassay) were determined at B, and during both T-LD and T-HD. LH profiles were analyzed by deconvolution and approximate entropy analyses. On T-LD, C and PCOS serum T levels increased 2- to 3-fold vs. B. On T-HD, T values doubled in both groups vs. T-LD. Controls on T-LD had greater 12-h pulsatile LH secretion rate (P < 0.05 vs. B) and on T-HD had lower mean, pulsatile, basal LH release and LH approximate entropy (vs. B, P < 0.05). PCOS did not respond to T-LD. High-dose T did not alter mean LH in PCOS but increased pulsatile and reduced basal LH secretion. PCOS adolescents have impaired suppression of pulsatile LH secretion rate consistent with reduced androgen negative feedback. Attenuation of T feedback in nonobese adolescents with PCOS extends the pathophysiology of this syndrome.

Obesity and the Emergence of Sleep-Wake Gonadotropin Secretion in Girls during Early Pubertal Development

It has been clearly established that obesity is associated with reduced gonadotropin secretion in men and women. In normal women, early studies involving relatively few subjects had shown that serum LH levels were significantly reduced in obese individuals compared with levels of normal-weight controls (1, 2). Subsequent larger studies that examined the relationship of LH secretory dynamics to body weight included women with polycystic ovary syndrome (PCOS) as well as normal women (3, 4). In particular, LH secretion in both groups of obese women was characterized by reduced basal levels and decreased pulse amplitude, whereas LH frequency appeared unaffected. Recently, it was demonstrated that morbidly obese women with normal menstrual function exhibited a similar pattern of LH suppression compared with that of normal women (5). In adolescent girls, there are essentially no data concerning the influence of obesity on the rate and amplitude of LH secretion. However, during puberty the progressive maturation of gonadotropin secretion in normal girls through various stages of development has been well documented (6). Given the observed impact of obesity on LH secretion in adults, it seems logical to investigate this relationship at a time when activation of hypothalamic-pituitary interaction is emerging. This notion is underscored by the association of obesity with an earlier onset of puberty and precocity, increased risk of chronic anovulation with menstrual irregularities, and greater likelihood of PCOS. The last of these conditions commonly manifests clinical symptomatology during or soon after puberty. In this issue of JCEM, McCartney et al. (7) have extended their efforts to define the role of obesity on gonadotropin secretion during pubertal development. In previous studies using a cross-sectional design, they showed that during early puberty (Tanner 1–3), obese girls had significantly increased serum total and free testosterone levels compared with nonobese controls accompanied by corresponding decreases of circulating LH concentrations (8, 9). In addition, obese girls exhibited hyperinsulinemia and increased homeostasis model of assessment values compared with that of normal-weight controls, which may have accounted, in part, for hyperandrogenemia in the presence of lowered serum LH. In early pubertal girls, diurnal variation was observed for both testosterone and progesterone with levels lower at night and rising in the early morning hours. The incremental change in both hormones was significant in nonobese girls but failed to achieve statistical significance in obese girls likely because of too few numbers. Nevertheless, the heightened level of progesterone in obese girls may have contributed to reduced LH levels in early puberty. This notion relates to the insensitivity of the GnRH pulse generator to progesterone negative feedback in women with PCOS. In normal women, progesterone administration inhibits LH secretion, e.g. pulse frequency. Prior studies by this group have shown that androgen may interfere with this feedback mechanism as suggested by reduction of rapid LH release in response to progesterone administration in women with PCOS treated with an antiandrogen, which implies that hyperandrogenemia may contribute to increased LH secretion in this disorder. This proposed mechanism is suggested in young adolescents because some girls with PCOS treated with progesterone failed to exhibit a reduced rate of LH release (10). Because their earlier studies had been restricted to basal serum LH measurements, McCartney et al. (7) proceeded to conduct an extensive frequent sampling study of obese [body mass index (BMI) 95th percentile] and nonobese (BMI 95th percentile) girls at Tanner stages 1–5 and found differences in LH secretion within and between groups during early puberty (11). The experimental design employed a sampling interval of 1900–2300 h to denote daytime secretion and 2300–0300 h and 0300– 0700 h to reflect nighttime release. As expected, LH pulse frequency, amplitude, and mean concentrations in nonobese Tanner 1–2 girls were, in general, greater with sleep than values observed in awake subjects. In Tanner 1–2 obese girls, there was little change in LH secretion over the same intervals of study.

Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals

Kisspeptins, the products of KiSS-1 gene, have recently emerged as fundamental regulators of reproductive function in different mammalian and, presumably, non-mammalian species. To date, a single form of KiSS-1 has been described in mammals, and recently, in several fish species and Xenopus. We report herein the cloning and characterization of two distinct KiSS-like genes, namely, KiSS-1 and KiSS-2, in the teleost sea bass. While KiSS-1 encodes a peptide identical to rodent kisspeptin-10, the predicted KiSS-2 decapeptide diverges at 4 amino acids ( FN FN PFGLR F). Genome database searches showed that both genes are present in non-placental vertebrate genomes. Indeed, phylogenetic and genome mapping analyses suggest that KiSS-1 and KiSS-2 are paralogous genes that originated by duplication of an ancestral gene, although KiSS-2 is lost in placental mammals. KiSS-1 and KiSS-2 mRNAs are present in brain and gonads of sea bass, medaka and zebrafish. Comparative functional studies demonstrated that KiSS-2 decapeptide was significantly more potent than KiSS-1 peptide in inducing LH and FSH secretion in sea bass. In contrast, KiSS-2 decapeptide only weakly elicited LH secretion in rats, whereas KiSS-1 peptide was maximally effective. Our data are the first to provide conclusive evidence for the existence of a second KiSS gene, KiSS-2, in non-placental vertebrates, whose product is likely to play a dominant stimulatory role in the regulation of the gonadotropic axis at least in teleosts.

Central Lipoprivation-Induced Suppression of Luteinizing Hormone Pulses Is Mediated by Paraventricular Catecholaminergic Inputs in Female Rats

The present study aims to clarify the role of fatty acids in regulating pulsatile LH secretion in rats. To produce an acute central lipoprivic condition, mercaptoacetate (MA), an inhibitor of fatty acids oxidation, was administered into the fourth cerebroventricle (4V) in ad libitum fed ovariectomized (OVX) rats (0.4, 2, and 10 micromol/rat) with or without an estradiol (E2) implant producing diestrus plasma E2 levels. Pulsatile LH secretion was suppressed by 4V MA administration in a dose-dependent manner in both OVX and OVX plus E2 rats. Mean LH levels and LH pulse frequency and amplitude were significantly reduced by the highest dose of MA in OVX rats, and by the middle and highest dose of MA in E2-treated rats, suggesting that estrogen enhanced LH suppression. Blood glucose levels increased immediately after the highest dose of MA in both groups. Fourth ventricular injection of trimetazidine (2 and 3 micromol/rat), another inhibitor of fatty acids oxidation, also inhibited pulsatile LH release, resulting in significant and dose-dependent suppression of LH pulse frequency and an increase in blood glucose levels in OVX plus E2 rats. In contrast, peripheral injection of the highest 4V dose of MA (10 micromol/rat) did not alter LH release or blood glucose levels. Microdialysis of the hypothalamic paraventricular nucleus (PVN) revealed that norepinephrine release in the region was increased by 4V MA administration. Preinjection of alpha-methyl-p-tyrosine, a catecholamine synthesis inhibitor, into the PVN completely blocked the lipoprivic inhibition of LH and the counter-regulatory increase in blood glucose levels in OVX plus E2 rats. Together, these studies indicate that fatty acid availability may be sensed by a central detector, located in the lower brainstem to maintain reproduction, and that noradrenergic inputs to the PVN mediate this lipoprivic-induced suppression of LH release.

Gonadotropin-releasing hormone neurons coexpress orexin 1 receptor immunoreactivity and receive direct contacts by orexin fibers.

The orexins are produced in neurons of the lateral hypothalamic area and implicated in the regulation of both feeding and reproductive function. Orexins stimulate LH secretion in steroid-primed ovariectomized female rats and suppress LH secretion in nonprimed ovariectomized rats. The aim of the present study was to characterize the neuroanatomical pathway by which orexin might modulate LH secretion in the rat. Using double- and triple-label immunofluorescence coupled with confocal microscopy, we found that 75-85% of GnRH neurons were contacted by orexin fibers, and triple labeling with synaptophysin provided additional confirmation of close contacts. Furthermore, about 85% of GnRH neurons were colocalized with the orexin receptor 1 (OX-R1), and the OX-R1-expressing GnRH neurons were contacted by orexin terminals, providing the basis for a functional neuroanatomical pathway. GnRH nerve terminals in the median eminence, however, do not express OX-R1. An additional study investigated the coexpression of neuropeptide Y Y4-like receptors and orexin fibers in relation to GnRH neurons. There is evidence that Y4 receptor stimulation results in LH release, and studies from our laboratory show Y4-like immunoreactivity in the majority of orexin cell bodies in the lateral hypothalamic area and some orexin fibers scattered throughout the hypothalamus. The present study found that, although Y4-positive orexin fibers are in present in the area of GnRH neurons, they never come in close contact with GnRH neurons. Together, these data suggest that Y4 receptor modulation of LH release is likely to be indirect through orexin cell bodies and that orexin modulates GnRH neurons directly via OX-R1.

Poorly controlled type I diabetes mellitus in young men selectively suppresses luteinizing hormone secretory burst mass.

Alterations in the reproductive axis function are present to a variable extent in patients with type 1 diabetes mellitus (IDDM). Results from studies in IDDM men have yielded discrepant findings, which may reflect nonuniform patient selection criteria, age, diabetic status, duration of the disease and differences in sampling protocols. To more clearly define the impact of early diabetic alterations in the male reproductive axis, we applied a combined strategy of patient selection restricted to young men with relatively short duration of IDDM, dual control groups, multiparameter deconvolution analysis to assess LH secretory activity, and assessment of time-dependent changes in human chorionic gonadotropin (hCG)-stimulated serum testosterone concentrations. Three groups of subjects were studied: 11 young men with poorly controlled IDDM, 9 well controlled diabetics, and 9 healthy men. All volunteers underwent blood sampling at 10-min intervals before and after 2 consecutive iv pulses of 10 micro g GnRH. On a separate day, 40 IU/kg hCG were given im, and blood samples were collected before hCG administration, every 60 min thereafter for 6 h, and then 24, 48, and 72 h after the injection. Mean serum LH concentrations across the basal 6-h sampling period were significantly (P < 0.05) decreased in men with poorly controlled IDDM (11 +/- 1.6 IU/liter) compared with those in well controlled diabetics (19 +/- 1.8 IU/liter) and healthy controls (19 +/- 1.5 IU/liter). Multiple parameter deconvolution analysis revealed a 50% reduction in the mass of LH secreted per burst and the pulsatile LH secretion rate in poorly controlled IDDM (mass of LH secreted/burst, 7 +/- 1.1 vs. 12 +/- 2.1 and 13 +/- 1.5 IU/liter; LH secretion rate, 47 +/- 6.3 vs. 78 +/- 10 and 87 +/- 11 IU/liter.6 h; poorly controlled vs. well controlled IDDM and healthy controls, respectively; P < 0.05 for both parameters). Uncontrolled IDDM patients had significantly (P < 0.05) lower integrated serum LH concentrations after the first and second GnRH pulses (first GnRH pulse, 4460 +/- 770 vs. 7250 +/- 1200 and 5120 +/- 910 IU/liter; second pulse, 4700 +/- 615 vs. 7640 +/- 881 and 7100 +/- 1230 IU/liter; poorly controlled vs. well controlled IDDM and healthy men, respectively) and markedly attenuated LH secretory burst mass after the second GnRH stimulus (49 +/- 8.8 vs. 90 +/- 13 and 83 +/- 19 IU/liter; poorly controlled vs. well controlled IDDM and healthy controls, respectively). The biological to immunological ratio of LH released in baseline conditions was higher in uncontrolled IDDM patients (0.81 +/- 0.10) than in controlled IDDM (0.37 +/- 0.08) and healthy controls (0.48 +/- 0.06; P < 0.01), whereas LH released in response to exogenous GnRH exhibited comparable ratios among the three study cohorts. Baseline serum testosterone levels as well as absolute and incremental responses to exogenous hCG did not differ by degree of metabolic control. Collectively, these results indicate that the function of the hypothalamic-gonadotrope axis is compromised in young men with poorly controlled IDDM, such that the amplitude of spontaneous pulsatile and exogenous GnRH-stimulated LH secretion is attenuated. This central hypogonadotropism is paradoxically associated with the presence in the circulation of gonadotropin molecules with enriched biological activity, which is evidently sufficient to temporarily maintain normal total testosterone concentrations in the earlier stages of IDDM.

Dissociation of early folding events from assembly of the human lutropin beta-subunit.

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common alpha-subunit and beta-subunits that confer biological specificity. LHbeta and CGbeta share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHbeta assembles with the alpha-subunit more slowly than does hCGbeta, and only a fraction of the LHbeta synthesized is secreted, whereas CGbeta is secreted efficiently. To understand why the assembly and secretion of these related beta-subunits differ, we studied the folding of LHbeta in CHO cells transfected with either the LHbeta gene alone, or in cells cotransfected with the gene expressing the common alpha-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pbeta1, to the second major folding form, pbeta2, did not differ significantly from the pbeta1-to-pbeta2 conversion of CGbeta, suggesting that variations between the intracellular fates of the two beta-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGbeta, where the folding to pbeta2 results in an assembly-competent product, apparently greater than 90% of the LH pbeta2 recovered from LHbeta-transfected CHO cells was assembly incompetent, accounting for inefficient LHbeta assembly with the alpha-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGbeta, all 12 LHbeta cysteine residues formed S-S linkages as soon as pbeta2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pbeta2 and excess urinary alpha-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGbeta, LHbeta homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective beta-subunits.

Effects of melatonin implants in pony mares 2. Long-term effects

The effects of melatonin implant treatment over a 4 wk period at the summer solstice on the transition into and out of the following anovulatory season were evaluated in ovary-intact and ovariectomized mares. Melatonin implants tended to delay the timing of the final ovulation of the breeding season (P=0.0797) in the ovary-intact mares. Although the decline in LH secretion associated with the end of the breeding season was parallel between treatments and ovarian statuses, the rate of LH secretion, as expressed by its mathematical accumulation,was lower in ovariectomized, melatonin-treated mares than in ovariectomized, control mares suggesting that melatonin administration advanced the offset of the breeding season in ovariectomized mares (P=0.0001). The first ovulation of the subsequent breeding season was significantly delayed in the melatonin-treated mares as compared with that of control mares (P=0.0031). During reproductive recrudescence, the time of the onset of the increase in LH secretion was similar among all 4 groups but the patterns of LH secretion were different for each treatment and ovarian status combination (P=0.0112). Mares with melatonin implants had a slower rate of increase in LH secretion than control mares (P=0.0001), and ovariectomized mares had a faster rate of LH increase than intact mares (P=0.0001). These results suggest that melatonin implants during the summer solstice can alter the annual reproductive rhythm in mares and support the concept that endocrine patterns of reproductive recrudescence are not entirely independent of the ovary.

Pulsatile intravenous gonadotropin-releasing hormone administration averts fasting-induced hypogonadotropism and hypoandrogenemia in healthy, normal weight men.

Fasting or severe caloric restriction in the human or experimental animal suppresses serum LH and sex steroid concentrations. In healthy men undergoing prolonged (5-day) nutrient deprivation, the daily LH secretion rate, the mass of LH secreted per burst, and the serum testosterone concentration fall markedly, with no decrease in responsiveness to a single bolus of GnRH. Here we test the hypothesis that the hypogonadotropic hypoandrogenemia accompanying fasting reflects decreased endogenous GnRH release. To this end, six healthy young men were studied on a fed day and during two 83-h fasting sessions with concurrent saline or pulsatile GnRH administration (100 ng/kg, i.v., every 90 min for 24 h) followed by a single bolus of 10 microg GnRH, i.v., to evaluate pituitary responsiveness. We employed a highly sensitive LH immunoradiometric assay, which correlates well with an in vitro Leydig cell bioassay, and deconvolution analysis to calculate in vivo LH secretory burst frequency, amplitude, duration, mass, and LH half-life. Fasting resulted in 30-50% declines in serum total and free testosterone and LH concentrations, and a 3-fold decrease in the calculated 24-h LH secretion rate (fed, 42 +/- 12; fasting, 14 +/- 1.9 U/L distribution volume x day; mean +/- SEM; P < 0.05, by ANOVA). Reduced LH secretion was accounted for by dual mechanisms, viz. a fall in both the apparent number of computer-resolved LH secretory bursts per 24 h (fed, 16 +/- 1.1; fasting, 10 +/- 1.2; P < 0.01) and the mass of LH secreted per burst (fed, 2.5 +/- 0.5; fasting, 1.5 +/- 0.1 U/L; P < 0.05). Fasting also decreased the mean value of the 24-h (nyctohemeral) rhythm in serum LH concentrations and reduced the approximate entropy (disorderliness) of LH release. Exogenous pulsatile GnRH injections prevented both the reduction in the calculated daily LH secretion rate (fed, 42 +/- 12; fasting plus GnRH, 64 +/- 16 IU/L; P = NS) and the decline in serum testosterone concentrations (fed, 556 +/- 71 ng/dL; fasting, 391 +/- 41; fasting plus GnRH, 859 +/- 65). Pulsatile GnRH treatment also restored the nyctohemeral mesor of serum LH concentrations and the approximate entropy value to baseline. Administration of a submaximal dose of exogenous GnRH (10 microg, i.v.) at the end of the fasting interval revealed statistically identical LH release in the three study groups, suggesting that pituitary responsiveness to GnRH was unchanged in this paradigm. In summary, a pulsatile iv GnRH infusion in young men averts completely the fasting-induced decline in LH secretory burst mass/amplitude and frequency, reinstates serum total and free testosterone concentrations, and restores the mesor of LH's nyctohemeral rhythmicity and the approximate entropy of LH release. Rescue of hypogonadism by pulsatile GnRH stimuli supports the thesis that nutrient withdrawal decreases the output of the human hypothalamic GnRH burst generator.