LH Increments Research Articles

The effects of kisspeptin agonist canine KP-10 and kisspeptin antagonist p271 on plasma LH concentrations during different stages of the estrous cycle and anestrus in the bitch

Kisspeptin (KP) plays a key role in the regulation of the hypothalamic-pituitary-gonadal axis via the release of GnRH. As normal KP signaling is essential for reproductive function, it could be an interesting new target for therapeutic interventions, e.g., nonsurgical contraception in dogs. The aims of the present study were to investigate the effect of KP-10 administration on plasma LH concentration in different stages of the reproductive cycle and to investigate the suitability of p271 as KP antagonist in the bitch. Two groups of six adult Beagle bitches were used. In one group, plasma LH concentration was determined before (40 and 0 minutes) and 10, 20, 40, and 60 minutes after the intravenous administration of 0.5-μg/kg body weight (BW) canine KP-10. In the other group, the bitches received a continuous intravenous infusion with p271 (50 μg/kg BW/h) for 3 hours, and 0.5-μg/kg BW canine KP-10 was administered intravenously 2 hours after the start of the p271 infusion. Their plasma LH concentration was determined before (−40 and 0 minutes) and 30, 60, 90, 120, 130, 140, 160, and 180 minutes after the start of the p271 infusion. In both groups, the experiments were performed during the follicular phase, the first and second half of the luteal phase, and during anestrus. Canine KP-10 induced an increase of plasma LH concentration during all estrous cycle stages and anestrus. There was no difference in LH response between the two groups. The lowest LH response was seen during the follicular phase and the highest response during anestrus. The area under the curve (AUC) for LH and LH increment in the follicular phase were lower than those in anestrus. The AUC LH and LH increment in the first half of the luteal phase were lower than those in the second half of the luteal phase and anestrus. The AUC LH and LH increment in the second half of the luteal phase were not different from those in anestrus. Continuous administration of the antagonist p271 did not alter basal plasma LH concentration and could not prevent or lower the LH response to KP-10 in any of the cycle stages and anestrus. It can be concluded that the LH response to KP-10 is dependent on estrous cycle stage and that peripheral administrated p271 cannot be used as KP antagonist in the dog. This provides new insight in reproductive endocrinology of the bitch, which is important when KP signaling is considered for therapeutic interventions, such as for estrus induction or nonsurgical contraception in the bitch.

Clinical value of triptorelin stimulation test in differential diagnosis of disorder due to delayed puberty: 128 cases report

Objective To evaluate the diagnostic value of triptorelin stimulation test in disorders due to delayed puberty.Methods Triptorelin stimulation test was carried out in 128 teenagers with delayed puberty,due to idiopathic hypogonadotropic hypogonadism(IHH) in 77 cases and constitutional delayed puberty(CDP) in 51.Blood samples were obtained 15 min before and 0,30,60,and 120 min after tripotorelin administration,and the levels of LH and FSH were determined.An extended GnRH stimulation test was carried out in 3 patients with IHH.Results Peak LH,peak FSH,and LH increment were parameters with high diagnostic value.A cut-off point at 8.2 IU/L of peak LH showed a sensitivity of 87％ and a specificity of 80.4％ in the differential diagnosis of IHH and CDP.Conclusion Peak LH cut-off point at 8.2 IU/L of triptorelin stimulation test seems to be sufficient to confirm diagnosis of IHH and CDP.An extended GnRH stimulation test may distinguish hypothalamic from the pituitary hypogonadotropic hypogonadism. Key words: Idiopathic hypogonadotropic hypogonadism; Constitutional delayed puberty; Triptorelin stimulation test ; LH

Knockdown of GABAA Receptor Signaling in GnRH Neurons Has Minimal Effects upon Fertility

The amino acid gamma-aminobutyric acid (GABA) is thought to play a key role in shaping the activity of the GnRH neurons throughout embryonic and postnatal life. However, the physiological roles of direct GABA inputs to GnRH neurons remain unknown. Using a Cre-LoxP strategy, we generated a targeted mouse line, in which all (98 +/- 1%) GnRH neurons had the gamma2-subunit of the GABA(A) receptor deleted. Electrophysiological recordings of GABA(A)-mediated postsynaptic currents from green fluorescent protein-tagged GnRH neurons with the gamma2-subunit knocked out (GnRH gamma2 KO) showed that the amplitude and frequency of GABA(A) postsynaptic currents were reduced by 70% (P < 0.01) and 77% (P < 0.05), respectively, and that the response to exogenous GABA was reduced by 90% (P < 0.01). Evaluation of male and female GnRH gamma2 KO mice revealed completely normal fecundity, estrous cycles, and puberty onset. Further investigation with gonadectomy and different steroid replacement regimens showed normal basal levels of LH in both sexes, and a normal estradiol-evoked positive feedback mechanism in females. However, the increment in LH after gonadectomy in GnRH gamma2 KO female mice was double that of controls (P < 0.05) and also more potently suppressed by 17-beta-estradiol (P < 0.05). A similar but nonsignificant trend was observed in GnRH gamma2 KO male mice. Together, these findings show that 70-90% reductions in the normal levels of GABA(A) receptor activity at the GnRH neuron appear to impact upon the estrogen negative feedback mechanism but are, nevertheless, compatible with normal fertility in mice.

Age in Men Does Not Determine Gonadotropin-Releasing Hormone’s Dose-Dependent Stimulation of Luteinizing Hormone Secretion under an Exogenous Testosterone Clamp

Aging is associated with a decline in incremental LH pulse amplitude, which could be due to decreased GnRH secretion or impaired GnRH action. Inconsistent published studies of GnRH action in older men may be due to disparate sex-steroid milieus. This study was conducted at a clinical translational-research unit. We studied 16 healthy men (8 young men and 8 older men). An overnight transdermal testosterone (T) clamp was implemented before randomly ordered injections of 0, 2.5, 10, 25, 250, and 750 ng GnRH on separate days (96 study sessions). LH responses were quantified by variable-waveform deconvolution analysis. The T clamp maintained age-invariant mean concentrations of total, bioavailable, and free T, SHBG, LH, FSH, and prolactin. By two-way analysis of covariance, GnRH dose (P < 0.001) but not age (0.15 < or = P < or = 0.83) determined mean, peak, incremental, and pulsatile LH responses. Statistical power (median) was 95, 98, 90, and 99% to detect a 30% or greater age contrast at P < or = 0.05 in mean, peak, incremental, and pulsatile LH responses, and greater than 99% to detect a 30% or greater age contrast in bioavailable or total T concentrations. Higher GnRH doses (P < 0.001) abbreviated LH secretory bursts in both age groups. In the face of eugonadal concentrations of total, bioavailable, and free T, young and older men exhibit remarkably similar LH responses to a 300-fold dose range of exogenous GnRH. Accordingly, previously reported disparate effects of age on GnRH action may reflect in part age-discrepant sex-steroid milieus.

Reproductive suppression and the seasonality of reproduction in the social Natal mole-rat ( Cryptomys hottentotus natalensis)

Natal mole-rats are social subterranean rodents which exhibit a reproductive division of labour. Reproduction is confined to a single breeding female and one or more males; the remainder of the colony members is reproductively suppressed by the presence of the breeding animals. Apart from the discovery that female Natal mole-rats are induced ovulators, little is known about the reproductive biology of this species. Natal mole-rats are closely related to common and highveld mole-rats, both of which are induced ovulators and seasonal breeders. We therefore postulated that reproduction in Natal mole-rats is seasonally regulated. However, the results indicate that dominant Natal mole-rats are able to reproduce in the winter as well as in the summer. Furthermore, the increment in plasma LH in response to GnRH does not show marked seasonal differences in reproductive or non-reproductive mole-rats of either sex. Nevertheless, in all reproductive categories the level of plasma LH is significantly higher in the winter than in the summer. Seasonality in plasma LH levels dissociated from seasonality in breeding seems paradoxical. Further investigations will be required to elucidate this finding. We also investigated the processes underlying socially regulated reproduction in this species by determining basal and GnRH-evoked plasma LH in reproductive and non-reproductive animals of each sex in both seasons. The results failed to identify neuroendocrine differences consistent with an inhibited reproductive state in subordinates of either sex. Thus, the present findings suggest that behavioural interactions and/or inbreeding avoidance are the principal factors underlying suppression of reproduction in subordinate Natal mole-rats.

Variations in pituitary-gonadal suppression during intranasal buserelin and intramuscular depot-triptorelin therapy for central precocious puberty. Belgian Study Group for Pediatric Endocrinology.

This study evaluated pubertal development, growth and pituitary-gonadal suppression in 21 patients with central precocious puberty treated with buserelin intranasally and switched after a mean of 2.1 yr to depot-triptorelin given im for 1 year. Arrest or regression of puberty was observed in 12 patients while progression of puberty during therapy was seen in 9 patients (6 on buserelin, 2 on triptorelin and 1 on both therapies). The increment in serum LH and FSH concentrations after sc injection of short-acting triptorelin was greater on buserelin than on triptorelin therapy, particularly in patients with evidence of progression of puberty. Height velocity during therapy showed a reduction which paralleled the decelerating phase of the normal pubertal growth spurt. The rate of bone maturation during therapy was inversely related to pretreatment bone age. Predicted final height showed marked individual variations which were inversely related to predicted adult height before therapy. These data indicate that differences in the nature and route of administration of Gn-RH agonist therapy for central precocious puberty can be of importance for inhibition of pituitary gonadotropin secretion and development of secondary sex characteristics. Height velocity and bone maturation are age-related and the change in predicted adult height depends on pretreatment level.

Reproductive Effects of Ethnomedicinal Formulation of Tape-Vine Leaves in Female Rats

Documented ethno-contraceptive use of Tape-vine or Stephania japonica (THUNB.) MIERS., Syn. Stephania hernandifolia (WILLD.) WALP. leaves is evaluated with regards to post-coital pregnancy interceptive activity of its aqueous extract (AE) and an ethnomedicinal formulation (EF) in Wistar rats. EF at 500 and 250 mg/kg doses induced 66.7% and 33.3% post-coital pregnancy interception respectively and the higher dose exhibited significant reduction in number of litters born and also anti-implantation property. In contrast, none of the dose levels of AE interfered in pregnancy but significant anti-implantation property was observed at doses of 2 and 1 g/kg, even as the higher dose produced significant reduction in number of litters born as well. EF at 500 mg/kg also exhibited significant uterotrophic activity and histological changes in uterus. Pair-wise comparison of sex hormone-levels exhibited significant increment in serum estradiol, LH and FSH but decrease in progesterone levels. Assessed blood lipid-carbohydrate profile exhibited substantial decrease in glucose, cholesterol, VLDL and triglyceride contents and significant increase in HDL. It is concluded that EF probably acts as better post-coital pregnancy interceptive agent through restriction of implantation by alteration of gonadal hormone levels and decline in blood-glucose levels that possibly disrupts oxidative energy metabolism in uterus during implantation. High surge in LH and FSH suggests negligible interference in ovulatory mechanism. This preparation also seems to be free of cardiovascular risk factors. HPTLC and HPLC analysis of both EF and AE exhibited marked chemical differences.

Pulsatile intravenous infusion of recombinant human luteinizing hormone under acute gonadotropin-releasing hormone receptor blockade reconstitutes testosterone secretion in young men.

The present study tests the hypothesis that iv infusion of discrete pulses of recombinant human (rh)LH after overnight GnRH-receptor blockade can restore midphysiological concentrations of testosterone (Te) in normal young men. In a pilot time-course analysis, injection of the GnRH antagonist ganirelix (2.0 mg sc) at 2200 h lowered LH concentrations (mean +/- sem) from 3.4 +/- 0.7 to 0.8 +/- 0.1 IU/liter (P < 0.01) and Te concentrations from 416 +/- 48 to 107 +/- 16 ng/dl (P < 0.01) (to convert to nmol/liter, multiply by 0.0347) at 0800 h the next morning. LH and Te concentrations remained suppressed thereafter for an additional 15 h (interval, 10-25 h after ganirelix administration) at mean values of 1.2 +/- 0.1 IU/liter and 67 +/- 10 ng/dl, respectively (P < 0.005 vs. baseline). Based on these data and earlier dose-finding studies, eight men received a single ganirelix injection followed by seven consecutive iv pulses of rhLH (15.3 IU Second International Reference Preparation) each delivered over 6 min every 2 h beginning at 0800 h. Recurrent rhLH stimuli restored mean LH concentrations (IU/liter of homologous standard) to 4.8 +/- 0.3, LH peak maxima to 7.1 +/- 0.6, incremental LH peak amplitudes to 3.7 +/- 0.4, and interpeak nadir LH concentrations to 3.3 +/- 0.3 (each P < 0.01 vs. saline infusion after ganirelix). These values were indistinguishable from the normal 95% range established in 23 young adults of comparable age. Injected LH pulses increased total Te concentrations (ng/dl) to 440 +/- 52, Te peak maxima to 552 +/- 64, incremental Te amplitudes to 188 +/- 23, and interpeak nadir Te concentrations to 366 +/- 43 (each P < 0.01 vs. saline addback; P value not significant vs. untreated men). Under combined ganirelix inhibition and pulsatile rhLH drive, Te concentrations rose from a nadir of less than 120 ng/dl to an asymptotic plateau of 611 ng/dl with an estimated half-time of 97 +/- 9.1 min. Cross-correlation analysis of paired serial LH and Te concentrations verified that infused LH pulses stimulate Te elevations within 40-70 (median 50) min (P < 0.001). Kinetic estimates of the half-life of exogenous rhLH averaged 107 +/- 3.8 min, which value exceeded that of secreted LH monitored after pharmacological GnRH stimulation (83 +/- 12 min; P = 0.012). We conclude that intermittent iv pulses of rhLH delivered over 12 h under selective GnRH-receptor blockade can restore young adult-like pulsatile LH and Te concentrations with an appropriate time delay coupling the lutropic stimulus to the steroidogenic response. Whether a comparable near-physiological paradigm can maintain human Leydig-cell testosterone production for a more extended interval is not known.

Relationship of GnRH-stimulated LH release to episodic LH secretion and baseline endocrine-metabolic measures in women with polycystic ovary syndrome.

In polycystic ovary syndrome (PCOS) inappropriate gonadotrophin secretion is characterized by increased pulse frequency and amplitude, elevated 24-h mean serum concentrations, and greater responses to GnRH. While the mechanism(s) responsible for this increased release of LH are not well understood, enhanced LH secretion has been attributed to increased pituitary sensitivity to GnRH and feedback influences from circulating steroid hormones. To address these considerations, we conducted a study to examine the relationships between GnRH-stimulated LH responses, episodic gonadotrophin secretion, and baseline measurements of endocrine-metabolic function in PCOS. Serum LH responses to sequential multidose GnRH administration and pulsatile gonadotrophin secretion were examined in 13 PCOS and 13 normal women. Serum LH, steroid hormone, insulin and glucose levels were determined in blood samples obtained during assessment of episodic gonadotrophin secretion and LH responses to GnRH stimulation. Each subject was studied on two consecutive days. On study day 1 each subject underwent frequent blood sampling every 10 min for 12 h. On study day 2 each received sequential doses of GnRH, 2 microg, 10 microg and 20 microg, administered intravenously at 4-h intervals over a continuous 12-h period. Serum LH responses following GnRH were markedly greater in PCOS compared to normal women, as expected. In individual PCOS, peak LH responses to GnRH were significantly correlated with corresponding basal LH and LH pulse amplitude, but not LH pulse frequency. In the PCOS group, LH responses were positively correlated with serum oestradiol (E2) and inversely related to body mass index (BMI). Between-group differences in LH responsiveness disappeared when controlling for serum testosterone (T) levels. These results indicate that the absolute LH increment following GnRH is largely dependent on baseline LH levels and may account for the well-documented difference in LH responsiveness between PCOS and normal women. That neither LH responses to GnRH nor LH pulse amplitude were correlated to LH pulse frequency suggests involvement of other factors along with GnRH to account for increased LH secretion in PCOS. In addition to E2 and BMI, serum testosterone appears to be, at least in part, responsible for differences in LH secretion and release between PCOS and normal women.

Effect of oxytocin on nitric oxide activity controlling gonadotropin secretion in humans.

Previously described inhibitory effects of the nitric oxide synthase (NOS) inhibitor L-NAME on luteinizing hormone-releasing hormone (LH-RH)-induced LH and follicle stimulating hormone (FSH) secretion in humans suggested modulation by nitric oxide (NO) of the gonadotropin-releasing action of LH-RH. In order to establish whether oxytocin (OT) participates in this regulatory mechanism, 10 normal men were treated with LH-RH (100 micro g as an i.v. bolus) given alone or in the presence of L-NAME (40 micro g kg-1 injected plus 50 micro g kg-1 infused i.v. for 60 min), OT (2 IU injected plus 4 IU infused i.v. for 60 min) or a combination of both drugs. The administration of OT was unable to change the gonadotropin responses to LH-RH. In contrast, L-NAME significantly reduced both FSH and LH increments induced by LH-RH. When L-NAME was given in the presence of OT, the LH and FSH responses to LH-RH were similar to those observed after the administration of LH-RH alone. These data suggest antagonistic actions of OT and L-NAME in the control of NOS activity in regulation of gonadotropin secretion induced by LH-RH.

Muting of androgen negative feedback unveils impoverished gonadotropin-releasing hormone/luteinizing hormone secretory reactivity in healthy older men.

Plasma bioavailable testosterone concentrations decline in healthy older men without a uniformly commensurate rise in serum LH concentrations, which disparity is consistent with a hypothesis of relative hypogonadotropism. Likewise, preserved gonadotrope responsiveness to exogenous GnRH stimulation, despite an attenuated amplitude of endogenous LH pulses, points to reduced hypothalamic GnRH feedforward signaling in aging males. To appraise GnRH/LH secretory reserve more directly in older men, we have compared daily LH secretion, driven by profound short-term blockade of androgen biosynthesis by oral ketoconazole administration, in nine young (ages, 18-32 yr) and seven older (ages, 60-73 yr) volunteers. The ability to unleash endogenous GnRH-driven LH secretion in response to acute testosterone withdrawal was quantitated by sampling blood every 10 min, for 24 h, followed by high-precision immunoradiometric assay. The resultant serum LH concentration profiles were analyzed by: 1) model-free discrete peak detection (Cluster) analysis; 2) the approximate entropy statistic to quantitate pattern regularity; and 3) 24-h rhythmic (cosinor) analysis. At baseline, mean and integrated (24-h) serum LH concentrations were similar in both age cohorts. However, Cluster analysis established an elevated LH peak frequency [18 +/- 0.86 (older) vs. 13 +/- 1.3 pulses/24 h (young), P = 0.0028] and a reduced incremental LH pulse area [37 +/- 6.9 (older) vs. 106 +/- 20 (young) IU/L x min, P = 0.016] in older men. Approximate entropy calculations also revealed more irregular LH release patterns in older men before intervention (P = 0.00089). Feedback stress, achieved by ketoconazole-induced androgen deprivation, unmasked further neuroregulatory defects in older volunteers, who failed to equivalently increase the: 1) mean (24-h) serum LH concentration [i.e. to 5.0 +/- 0.99 (older men) vs. 9.0 +/- 1.1 (young) IU/L, P = 0.000071]; 2) maximal LH peak height (to 6.1 +/- 1.1 vs. 10.4 +/- 1.2 IU/L, P = 0.00043); 3) incremental LH pulse area (to 41 +/- 8.8 vs. 87 +/- 20 IU/L x min, P = 0.016); 4) interpeak nadir serum LH concentration (to 4.0 +/- 0.77 vs. 7.9 +/- 1.0 IU/L, P < 10(-6)); 5) the quantitable irregularity of LH release (P = 0.00089); and 6) the mesor of 24-h rhythmic LH secretion (P = 0.000062). In summary, experimental imposition of a novel hypoandrogenemic open-loop feedback stressor, for 48 h, to heighten hypothalamic GnRH feedforward drive, unveils impoverished augmentation of LH pulse mass, impaired orderliness of LH release, and diminished 24-h rhythmic LH secretion in older men. The foregoing trilogy of neuroregulatory defects identifies unequivocally attenuated hypothalamo-pituitary reactivity to muting of androgen negative feedback in the aging male.

GnRH and hCG tests in healthy adolescents and adults.

With the aim of obtaining reference values of GnRH test and of hCG test in a Venezuelan population, the authors evaluated the response of the pituitary LH and FSH to the GnRH action in 48 adolescents and 43 adults. Also, in 22 adolescents and in 22 adults from the same sample they evaluated the hCG effect on the gonadal steroids secretion. The maximum LH increment was obtained 30 min after GnRH stimulation, independently from the patients' age and their sexual development. FSH maximum increment was obtained 60 min after GnRH in the adults; meanwhile, the FSH maximum increment was obtained 30 min after GnRH stimulation in the Tanner stage 4 adolescents, and 90 min post-GnRH in the Tanner 5 adolescents. The gonadal steroid secretion 2, 24, 48, and 72 h post-hCG was significantly lower in the Tanner 4 adolescents. The gonadal steroid response post-hGC stimulation in the Tanner 5 adolescent group was similar to the one obtained in the adults. In late puberty the LH response to GnRH stimulus is not related either to age or to sexual development, contrary to the FSH response obtained after GnRH and the gonadal steroid response after hCG stimulus, both of which are related to age and patients' sexual development.

A randomized controlled study comparing the endocrine effects of pulsatile intravenous gonadotropin-releasing hormone after gonadotropin-releasing hormone agonist pretreatment versus clomiphene citrate in patients with polycystic ovary syndrome

Objective: To compare the endocrine changes in the follicular phase in infertile patients with polycystic ovary syndrome (PCOS) treated with either pulsatile IV GnRH after GnRH-agonist (GnRH-a) down-regulation or clomiphene citrate (CC). Design: Subgroup analysis within a randomized, controlled, multicenter study. Setting: Women referred to the Infertility Clinic, Catharina Hospital Eindhoven, The Netherlands. Patient(s): Twenty-eight infertile patients with PCOS. Intervention(s): Patients were randomly assigned to pulsatile IV GnRH (10–20 μg/90 min) after GnRH-a down-regulation or CC (50–150 mg; cycle days 3–7). Patients were monitored on alternate days with ovarian sonography and serum concentrations of E 2, LH, and FSH. Serum P and sonography confirmed ovulation. Main Outcome Measure(s): Serum concentrations of E 2, LH, and FSH were assessed before treatment was started and after each ovarian sonography. Result(s): In ovulatory cycles, no statistically significant differences were observed during the follicular phase comparing serum concentrations of E 2, LH, and FSH between the treatments. However, a significant increase in LH occurred in the GnRH group. Conclusion(s): No significant endocrine differences were observed between GnRH and CC treatment. However, there was a significant increase in the serum LH concentration during the follicular phase in the GnRH group. Increments of LH in the GnRH group may be due to recovery of endogenous LH secretion.

Evaluación del efecto agudo de la administración de GnRH sobre la secreción de leptina en mujeres sanas e hiperandrogénicas.

Several studies suggest that leptin modulates the reproductive axis function. Leptin may stimulate release of GnRH from hypothalamus and of gonadotrophins from the pituitary. A synchronicity of LH and leptin pulses has been described in healthy women and in patients with polycystic ovarian syndrome (PCOS), suggesting a relationship between the episodic secretion of LH and leptin. In vitro experimental studies have demonstrated that leptin administration promotes GnRH-LH release. However it is not established whether GnRH promotes the episodic secretion of leptin. To assess the response of LH and leptin to the administration of a GnRH bolus in hyperandrogenic and healthy women. Eleven hyperandrogenic and eleven healthy women of similar age and body mass index (BMI) were studied. Under basal conditions three blood samples were collected every 30 min before and after the administration of a GnRH bolus (100 micrograms). LH and leptin concentrations were measured in all samples. Testosterone, SHBG and estradiol were determined in the first sample. For data analysis, the increment of LH and leptin between 0-30 and 0-60 min was calculated. The LH and leptin areas under the curve (AUC) before and after GnRH administration were also calculated in both groups. After GnRH administration an increment in LH concentrations was observed in both groups; however, leptin concentrations were not modified. In both groups LH area under the curve increased after GnRH administration; however, the leptin area was not modified. These results suggest that circulating leptin concentration is not modulated by GnRH-LH.

Inappropriate gonadotropin secretion in polycystic ovary syndrome: influence of adiposity.

In recent years, there has been uncertainty concerning the association of inappropriate gonadotropin secretion (high LH and normal FSH) and the polycystic ovary syndrome (PCOS). In the present study, we ascertained the influence of body composition on LH pulsatile parameters in 33 PCOS and 32 normal cycling (NC) women across a wide range of body mass index (BMI, 19-42 kg/m2). Twenty four-hour pulsatile parameters for serum LH (10-min sampling) and pituitary gonadotropin responses to i.v. bolus GnRH (10 micrograms) were evaluated. Fasting (0800 h) FSH and steroid hormone concentrations and 24-h mean insulin levels were determined. Insulin sensitivity (SI) was assessed by rapid i.v. glucose tolerance test in a subset of 28 PCOS and 29 NC subjects. Our results showed that BMI, an indicator of relative adiposity, had a significant negative impact on 24-h mean LH pulse amplitude (r = -0.63, P < 0.001) and the peak increment of LH in response to GnRH stimulation (r = -0.41; P = 0.02) for PCOS but not NC women. In contrast, 24-h LH pulse frequency was uniformly increased (40%) in PCOS as compared with NC women independent of BMI. In PCOS women, the blunting of pulse amplitude with increasing BMI resulted in a decline in 24-h mean LH levels (r = -0.63, P < 0.001) and the ratio of LH/FSH (r = -0.44, P = 0.02) not seen in NC. With BMI < 30 kg/m2, 24-h mean LH values for PCOS women were greater than the normal range for NC in 95% (18/19) of cases, whereas 24-h LH levels failed to discriminate PCOS from NC women in 43% (6/14) of obese (BMI > 30 kg/m2) PCOS women. Thus, the diagnostic value of LH determinations is retained for PCOS women with BMI < 30 kg/m2. For screening purposes, the mean of two LH values in samples collected at 30-min intervals was found to have a discriminatory power equal to that of the 24-h mean. These findings suggest that 1) BMI negatively influences LH pulse amplitude in PCOS women principally by an effect at the pituitary level; 2) accelerated LH pulse frequency in PCOS women is not influenced by BMI and represents a basic component of hypothalamic dysfunction in PCOS women; and 3) BMI does not influence gonadotropin secretion in normal cycling women. Thus assessments of basal LH levels and the LH/FSH ratio in hyperandrogenic anovulatory women are clinically meaningful when BMI is taken into account. Investigations to define the factor(s) that link adiposity and the attenuation of LH pulse amplitude in PCOS women would add further understanding of this complex neuroendocrine-metabolic disorder.

Influence of melatonin and oestradiol on the opioidergic regulation of LH and prolactin release in pony mares.

The aim of this study was to investigate the influence of oestradiol, melatonin and season on the opioid regulation of LH and prolactin release. Effects of the opioid antagonist naloxone (0.5 mg/kg) on LH and prolactin secretion were determined in ovariectomized pony mares. In experiment 1, mares in January (n = 6) were pretreated with oestradiol benzoate (5 micrograms/kg) for 20 days. In experiment 2, beginning in May, mares (n = 7) received melatonin (15 mg) for 15 days and subsequently a combination of melatonin plus oestradiol for 20 days. In experiment 3, beginning in May, mares (n = 6) were pretreated with oestradiol for 30 days, left untreated for 12 days and then given melatonin for 35 days. In all experiments the animals were injected with the opioid antagonist naloxone and saline on 2 consecutive days prior to treatment. In experiment 1, animals received naloxone and saline on days 10 and 11 and 20 and 21 following oestradiol treatment. In experiment 2, naloxone and saline were administered on days 15 and 16 following melatonin treatment and on days 10 and 11 and 20 and 21 of melatonin plus oestradiol treatment. In experiment 3, the animals received naloxone and saline on days 10 and 11, 20 and 21 and 30 and 31 of oestradiol treatment, prior to melatonin treatment and on days 15 and 16, 25 and 26 and 35 and 36 following melatonin. In January (experiment 1), naloxone evoked a significant (P < 0.05) LH release at all times, however the LH increment in response to naloxone increased during oestradiol pretreatment (P < 0.05). During the breeding season (experiments 2 and 3), naloxone induced a significant (P < 0.05) increase in plasma LH concentrations when mares had not been pretreated with oestradiol or melatonin and after oestradiol pretreatment. Basal LH concentrations and the LH increment in response to naloxone increased significantly (P < 0.05) during the 30-day oestradiol pretreatment. Melatonin decreased the naloxone-induced LH release and the LH release in response to naloxone and saline no longer differed after 25 and 35 days of melatonin pretreatment. When melatonin was given together with oestradiol for 20 days, again a significant (P < 0.05) LH release in response to naloxone occurred. Prolactin release was significantly (P < 0.05) increased by naloxone when mares had been pretreated with only melatonin. The opioid antagonist did not affect prolactin release in mares that had not been pretreated or received oestradiol either alone or in combination with melatonin. In conclusion, in long-term ovariectomized mares, opioids inhibit LH secretion independent from ovarian factors. This opioid inhibition of LH secretion is enhanced by oestradiol and reduced by melatonin. Although short-term melatonin treatment inactivates the opioid regulation of LH release, a prolonged influence of melatonin as occurs in winter does not prevent activation of the opioid system. This indicates that effects of melatonin on the opioid regulation of LH release change with time. An opioid inhibition of prolactin secretion is activated by melatonin given for 15-35 days but is lost under the prolonged influence of a short-day melatonin signal in winter.

Disparate serum free testosterone concentrations and degrees of hypothalamo-pituitary-luteinizing hormone suppression are achieved by continuous versus pulsatile intravenous androgen replacement in men: a clinical experimental model of ketoconazole-induced reversible hypoandrogenemia with controlled testosterone add-back.

To investigate the neuroendocrine mechanisms underlying the negative feedback actions of testosterone on both the pulsatile mode of LH release and the entropy or disorderliness of the LH release process, we blocked testicular androgen biosynthesis using oral high dose ketoconazole treatment with concomitant low dose glucocorticoid replacement for 48 h in six healthy young men. Volunteers were then infused iv with saline or a total of 8.0 mg testosterone base over the second 24 h via either a continuous or a pulsatile (90-min boluses) delivery pattern. Discrete peak detection (Cluster analysis) was applied to obtain a model-independent estimate of the frequency of serum LH concentration peaks, maximal and incremental LH peak amplitudes, peak area, and interpeak nadir serum LH concentrations. Approximate entropy was used to quantify the relative orderliness/disorderliness of the LH release process over 24 h. Ketoconazole treatment markedly lowered 24-h mean serum total and free testosterone concentrations (by 17- and 9-fold respectively), and significantly increased LH pulse frequency, maximal LH peak height, and interpeak nadir serum LH concentrations. Continuous iv testosterone add-back increased 24-h pooled serum free testosterone concentrations 3-fold more and concomitantly reduced mean (24-h) serum LH concentrations by at least 2-fold more than pulsatile delivery of the same total daily amount of androgen. Both modes of testosterone infusion suppressed pulsatile LH release, but the effects were distinguishable; namely, treatment with continuous vs. intermittent androgen add-back, respectively, decreased LH pulse frequency and incremental LH pulse amplitude. Ketoconazole treatment alone also significantly increased approximate entropy values, indicating greater disorderliness of LH release during androgen removal. Approximate entropy/orderliness was restored to baseline by continuous, but not pulsatile, iv testosterone replacement. In conclusion, the present novel testosterone add-back clinical experimental paradigm indicates that 1) remarkably different 24-h mean serum free testosterone concentrations can result from continuous vs. pulsatile testosterone delivery into the bloodstream; 2) androgen negative feedback can exert frequency- as well as amplitude-dependent suppression of pulsatile LH release; and 3) testosterone is required to maintain an orderly 24-h LH release process in young men.

Post-menopausal and chronological age have divergent effects on pituitary and hypothalamic function in episodic gonadotrophin secretion.

Ageing is known to reduce gonadotrophin secretion in post-menopausal women. To what extent the hypothalamus and pituitary are involved in this process is not clear. The aim of this study was to compare pulse characteristics of FSH and LH in relation to chronological and post-menopausal age. Base on the gonadotrophin to GnRH, we assessed the extent to which pituitary and/or hypothalamic ageing is responsible for the observed changes. Blood samples were obtained from post-menopausal women every 10 minutes for 6 hours. Subsequently, 100 micrograms GnRH was administered intravenously and blood samples taken after 30, 60 and 90 minutes. Twenty healthy women aged 47-72 years and between 1 and 30 years after the menopause. Plasma LH and FSH were measured by immunoradiometric assay. Pulses were identified by a computerized pulse detection program. End points were the mean number and amplitude of pulses, the mean LH and FSH concentrations during the 6-hour study period and the maximal LH and FSH increments following GnRH. Mean LH and FSH levels did not change with chronological age but the LH pulse frequency declined significantly and the response to GnRH increased. Mean LH levels declined with post-menopausal age without alteration in LH pulse frequency but with a significant decrease in pituitary LH response to GnRH. FSH levels remained unchanged. Post-menopausal ageing seems to have a major suppressive effect on pituitary gonadotroph function, while chronological ageing mainly affects the hypothalamic regulation of LH secretion.

Age-related decrease in the opioid control of LH secretion during reproductive years in normal women.

Our previous studies showed that naloxone is unable to stimulate LH secretion in elderly men, suggesting a loss in the endogenous opioid inhibitory control of LH in senescence. In the present study, we examined whether increasing age during the reproductive period in women is associated with alterations in the LH-releasing effect of naloxone. Studies were performed in younger (age 20-28 years, n = 8) and older (age 40-48 years, n = 8) subjects with normal menstrual cycles and normal gonadal steroid levels to avoid the interference of premenopause or menopause on gonadotropin secretion. The LH response to naloxone (4 mg as an i.v. bolus plus 10 mg infused in 2 h) was tested not only in normal conditions, but also after chronic dopaminergic stimulation with bromocriptine (5 mg/day for 7 days), because this treatment has been found able to restore normal LH responses to naloxone in elderly men. All tests were performed on the 22nd day of normal menstrual cycles. Naloxone induced a 100% increase in plasma LH levels in the younger group. In contrast, naloxone enhanced only by 50% LH secretion in the older subjects. When experiments were repeated after bromocriptine treatment, the effect of naloxone did not change in the younger subjects, whereas it was significantly higher in the older group. In the presence of bromocriptine, naloxone-induced LH increment in the older group was indistinguishable from that observed in the younger group. These data suggest that during the reproductive period, increasing age is associated with an impairment in the endogenous opioid control of LH secretion. In addition, age-related dopaminergic alterations independent of circulating gonadal steroid levels appear to underlie the defective endogenous opioid control of LH secretion in normally cycling women.

'Hamster-like' cycles in testicular size in the absence of gonadotrophin secretion in HPD rams exposed to long-term changes in photoperiod and treatment with melatonin.

Long term changes in the secretion of FSH, LH and testosterone, and the size of the testis were measured in groups of hypothalamo-pituitary disconnected Soay rams (HPD rams, n = 8) and control Soay rams (HPD-sham operated and unoperated, total n = 8) while exposed to an artificial lighting regimen of alternating 16-weekly periods of long days (16L: 8D) and short days (8L: 16D), and when treated with a constant-release implant of melatonin given under long days (total study: 136 weeks). Short term provocation tests using NMDA (glutamate receptor agonist), GnRH and LH were used to assess functionality of the hypothalamus, pituitary gland and testis, respectively. Control rams expressed normal photoperiod-induced cycles in the reproductive axis. Blood concentrations of FSH, LH and testosterone were significantly increased under short days, and decreased under long days with parallel changes in testicular diameter. Treatment with implants of melatonin under long days mimicked the effect of short days and induced rapid reactivation of the reproductive axis. In the HPD rams the blood concentrations of FSH, LH and testosterone declined immediately after the HPD surgery and values remained close to the lower limit of detection of the radioimmunoassays throughout the experiment. LH pulses were absent in the HPD rams and NMDA failed to induce LH secretion consistent with functional disconnection of the pituitary gland from the hypothalamus. The testes regressed to a significantly smaller size in the HPD rams compared with controls even at the nadir of the sexual cycle (testis diameter: 30.2 +/- 0.7 vs 41.3 +/- 0.8 mm, HPD vs control rams, respectively). A low amplitude cycle in testicular diameter (peak to nadir: 5.0 +/- 0.7 mm) persisted in the HPD rams with a temporal pattern opposite to the controls (growth under long days instead of short days; 'hamster like'). In the HPD rams, the treatment with melatonin blocked the long day-associated increase in testicular size, without effects on FSH, LH and testosterone secretion, pituitary responsiveness to GnRH (LH increment) or testicular responsiveness to LH (testosterone increment). This was in contrast to the cyclical changes in all parameters induced by melatonin in the control rams. At post-mortem, the reproductive tract in HPD rams was markedly regressed compared with the controls. The efficiency of spermatogenesis was reduced with few germ cells maturing beyond primary spermatocytes. Immunocytochemical staining, however, revealed the maintenance of androgen receptor expression in Sertoli cells, pertibular cells and Leydig cells, and steroid activity as measured by 17 alpha-hydroxylase expression in Leydig cells. Overall, the absence of photoperiod-induced changes in gonadotrophin secretion in the HPD rams illustrates the dependence on regulation by the hypothalamus, presumably through the secretion of GnRH. The residual cycle in the size of the testes in the HPD rams was closely correlated with the photoperiod-induced changes in prolactin secretion which persisted in these animals (summary of previous published data included). The combined results support the view that melatonin acts in the hypothalamus to mediate effects of photoperiod on gonadotrophin secretion and in the pituitary gland to mediate effects on prolactin secretion (dual site hypothesis), and that FSH, LH and prolactin act synergistically to regulate the long-term cycle in testicular activity in the ram.