Luteinizing hormone secretion by male rat pituitary cells perifused in vitro: effect of experimental left varicocele and orchiectomy

It has been shown previously that experimental left varicocele in the rat, results in a bilateral decrease in intratesticular testosterone. In the present work, pituitary responsiveness to GnRH as a possible mediator of this effect has been examined. Unilateral varicoceles were created in adult rats. A second group of animals underwent a sham operation and a third underwent bilateral orchiectomy. Thirty days after surgery, rats from all three groups were sacrificed and their pituitaries were removed. Dispersed pituitary cells were perifused in Bio-Gel columns with varying concentrations of GnRH. The concentration of LH in the collected eluent was determined by radioimmunoassay. The mean, overall GnRH-stimulated LH immunoreactive secretion rate (ng/min/10(7) cells) by pituitary cells from rats with varicocele (0.062 +/- 0.11) was no different from the overall release from the sham-operated controls (0.051 +/- 0.007). The dose-response curves for GnRH-stimulated release of LH by dispersed pituitary cells in the two groups also were not different. The overall GnRH-stimulated LH release by cells from the orchiectomized rats (0.171 +/- 0.032) was significantly greater than release by cells from the sham-operated and varicocele rats, and the concentration-response curve from the orchiectomy group was significantly elevated over those of the other two groups. These results indicate that GnRH-stimulated immunoreactive LH release is not altered in rats with experimental left varicocele and, thus, is not the source of an endocrinopathy that leads to decreased intratesticular testosterone concentrations in these animals.     

Testosterone effects on luteinizing hormone and follicle-stimulating hormone responses to gonadotropin-releasing hormone in the mouse

Studies in the mouse have demonstrated for the first time in vivo regulation of gonadotropin-releasing hormone (GnRH) on the minute-to-minute dynamics of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release and the effects of testosterone on this regulation. Intact and castrated mice with different testosterone levels (3-9 ng/mL) were challenged with exogenous GnRH while under general anesthesia to block endogenous GnRH release. Plasma concentrations of LH and FSH were determined by radioimmunoassay from sequential blood samples collected from anesthetized mice with in-dwelling catheters. The release of LH was correlated with the infusion of different doses of GnRH (0.35, 3.5, and 35 ng) in both intact and castrated mice (r = 0.942, approximately 0.999). GnRH-stimulated LH release was significantly lower in intact mice and in castrated mice with high testosterone levels than in castrated mice with low testosterone levels (P < .05). However, GnRH did not induce FSH release except in castrated males with low testosterone levels and at the highest dose of GnRH. The profiles of FSH release in intact mice and castrated mice with the highest testosterone levels were significant lower than the other groups (P < .05). In conclusion, release of LH, but not FSH, was correlated with increasing dosages of GnRH (r = 0.970), and testosterone significantly suppressed GnRH-stimulated LH release in the mouse (P < .05).     

The pattern of LH release in the adult ram influences the testicular steroidogenic response to individual LH pulses and is regulated by testosterone negative feedback

Two experiments were conducted with adult intact rams (approximately 58 kg in body weight) in the nonbreeding season to investigate interrelationships between LH and testosterone secretion. In Experiment 1, LH pulse frequency was increased from approximately two to six peaks per 8 hours (for 56 hours) by injecting (iv) 10 micrograms NIH-LH-S18 every 80 minutes. Induction of a breeding season peak frequency produced a progressive 3-fold increase (P less than 0.01) in mean serum testosterone levels to values during the last 8 hours of treatment (12.6 +/- 1.2 ng/ml) that were 50% of those in the fall. In response to LH pulsing, testosterone peak amplitude increased (P less than 0.05) from 3.5 +/- 0.8 ng/ml to 6.7 +/- 0.7 ng/ml. In Experiment 2, the mean testosterone level was increased to breeding season values (for 96 hours) by injecting (im) 5 mg testosterone every 4 hours. Mean LH levels and LH peak frequency were decreased (approximately 70%, P less than 0.01) following 36 hours of treatment, and the LH response to exogenous GnRH was decreased (approximately 45%, P less than 0.01) by the final 4 hours Results indicate that for rams in the nonbreeding season, the testicular steroidogenic response to individual LH pulses is enhanced when pulse frequency is increased. When blood testosterone is elevated to breeding season levels, LH pulse frequency is severely impaired, while pituitary responsiveness to GnRH is diminished, as in the fall.     

Deconvolution analysis of bioassayable LH secretion and half-life in men with idiopathic oligoasthenospermia

To further investigate the nature of neuroendocrine disturbances of the hypothalamo-pituitary-gonadal axis in idiopathic male infertility, we studied 12 infertile men with oligoasthenozoospermia and 13 euspermic controls, matched for age and body mass index, by blood withdrawal at 10-min intervals for 8 h to analyse pulsatile release of bioactive LH (b-LH). The rat interstitial cell testosterone (RICT) bioassay was used in conjunction with a recently validated multiparameter deconvolution algorithm, to estimate the endogenous half-life of b-LH, its secretory burst frequency, amplitude, duration and mass. Oligoasthenospermic men exhibited significant ( p < 0.05) alterations within the LH axis; namely: (1) a prolonged half-life of b-LH (92 min in euspermic men, 127 min in oligoasthenospermic men); (2) a reduced b-LH secretory burst amplitude (2.2 ± 1.2 IU/l/min in euspermic men, 1.7 ± 0.8 IU/l/min in oligoasthenospermic men); (3) a lower bioactive/immunoactive (b/i) ratio for LH secretory burst amplitude (14 in euspermic men, 4 in oligoasthenospermic men); (4) a reduced b/i ratio in the mass of LH secreted per burst (5.4 in euspermic men, 4.1 in oligoasthenospermic men) and (5) decreased coordinate release of b-LH and testosterone in infertile men, as assessed by cross-correlation analysis. These disturbances differ from the neuroendocrine dysregulation described in other states of male hypogonadotrophism.     

Effects of short-term stimulation of serotoninergic pathways on the pulsatile secretion of luteinizing hormone in the absence and presence of acute opiate-receptor blockage

To investigate the role of the serotoninergic system in regulating pulsatile gonadotropin secretion in man, we tested the influences of a novel selective serotonin re-uptake inhibitor (fluoxetine HCl) on episodic LH release in men. Spontaneous LH pulsatility was assessed by computerized analysis of serial LH concentrations measured in blood samples withdrawn at 10 min intervals for 24 h. Possible alterations in pituitary responsiveness were tested by administering three consecutive two-hourly intravenous pulses of GnRH (10 micrograms, 10 micrograms, and 100 micrograms). The effects of fluoxetine (20 mg orally three times daily for one wk) were assessed in a double-blind, placebo-controlled design. Compared with the placebo, fluoxetine elicited no changes in 24 h mean serum LH concentrations, LH pulse characteristics (Cluster analysis), or LH secretion and clearance parameters assessed in response to exogenous GnRH administration (deconvolution analysis) in the presence of normal opiatergic tone (nine healthy young men), and during acute blockade of the opiatergic system (seven young men treated with the mu-opiate receptor antagonist, naltrexone). In summary, a selective enhancer of serotoninergic activity (fluoxetine HCl) does not affect pulsatile LH release basally or in the presence of acute inhibitory opiatergic tone. Since this probe does modify prolactin secretion in man, we conclude that stimulation of the serotoninergic system by this selective neuroendocrine probe shows no demonstrable coupling between the serotoninergic and the opiatergic pathways that modulate pulsatile LH release in man.     

Specific regulatory actions of dihydrotestosterone and estradiol on the dynamics of FSH secretion and clearance in humans

The authors investigated immunoactive and bioactive follicle-stimulating hormone (FSH) secretion and clearance in six healthy young men during steady-state infusions of vehicle (basal, B, 28 hours), dihydrotestosterone (DHT, 4.5 days), or estradiol (4.5 days) accompanied by blood sampling at 10-minute intervals for 28 hours. Serum FSH concentrations were assayed by a two-site immunoradiometric assay (IRMA) and two separate in vitro bioassays (rat granulosa and Sertoli cell systems). FSH measurements included: 24-hour mean serum concentrations (IRMA and bioassay), multiple-parameter deconvolution of 24-hour pulsatile FSH time series and FSH release in response to exogenous gonadotropin-releasing hormone (GnRH) boluses (IRMA) to assess secretion and clearance, and circadian serum FSH concentration rhythms by cosinor analysis (IRMA). We found: 1) a significant decrease in 24-hour mean IRMA FSH concentrations during DHT infusion while both in vitro estimates of FSH bioactivity were unchanged; 2) significant decreases in the mass of IRMA FSH secreted per 24 hours during DHT infusion; 3) significant decreases in the IRMA FSH half-life during estradiol infusion without any change in FSH interpulse interval; 4) no steroidal effects on FSH secretory responses to exogenous GnRH; and 5) abolition of basal circadian FSH rhythms during sex-steroid infusions. Based on these findings, we conclude that steady-state sex-steroid hormone infusions selectively alter IRMA FSH secretion and clearance without affecting IRMA FSH pulse frequency or mean concentrations of bioactive FSH.     

Spectrum of the pulsatile characteristics of LH release in normal men

To assess the spectrum of LH pulse characteristics in normal men, blood samples from 36 individuals were drawn at 20-minute intervals for 8 hours. The subsequent immunoactive LH concentrations were analyzed by computer algorithms to delineate the frequency and amplitude characteristics of pulsatile LH secretion. The absolute range for LH pulse frequency estimated by a modified threshold method was 1-6 pulses/8 hr, with a mean (+/- SEM) of 3.36 +/- 0.17 (median -3)pulses/8 hr. The distribution differed significantly from a Gaussian pattern. The mean LH pulse amplitude expressed as a percent increase above nadir was 92.1 +/- 6.1 (median-91.5%). When LH pulse amplitude was defined as an increment (mIU/ml) above nadir, the mean value was 5.13 +/- 0.4 (median -4.8) mIU/ml. These two expressions of amplitude were positively correlated (P less than 0.01), while the incremental (mIU/ml) pulse amplitude correlated inversely with pulse frequency (P less than 0.01). To examine the influence of more intensified rates of venous sampling on the spectrum of LH pulse properties, blood was sampled at 4-minute intervals for 8 hours in a subgroup of 13 men. Under these conditions, estimated LH pulse frequency was significantly higher, with a mean of 10.31 +/- 1.87 (median -9) pulses/8 hr compared with 20-minute sampling in the same individuals (P less than 0.001). Although the estimates of LH pulse frequency at 4-minute and 20-minute sampling intervals were significantly correlated (P less than 0.01), the dispersion of the LH pulse frequency estimates was considerably larger at more rapid rates of sampling. There was an absolute range of 2-20 pulses/8 hr for the 4-minute sampling, and 1-6 pulses/8 hr for the 20-minute sampling in the same individuals. This increase in LH pulse frequency and the broader dispersion of the range of frequencies estimated at 4-minute compared with 20-minute sampling intervals were confirmed using either another pulse detection algorithm, or separate criteria designed to adjust false-positive error rates in relation to sampling intensity. It was concluded that eugonadal men exhibit a broad spectrum of pulsatile LH characteristics, and the range of LH pulse attributes is even greater at more intensive rates of venous sampling. The results of this study in normal men demonstrate that a wider dispersion of physiologic LH pulse characteristics must be recognized in man.(ABSTRACT TRUNCATED AT 400 WORDS)     

Appraising the instantaneous secretory rates of luteinizing hormone and testosterone in response to selective mu opiate receptor blockade in late pubertal boys

The pulsatile properties of gonadotropin and testosterone release were examined before and after chronic mu opiate receptor blockade with naltrexone, 50 mg every other day, in four normal boys in late puberty (ages 14 8/12 to 15 1/12 years). The nature of spontaneous secretory events was appraised for immunoactive LH and testosterone in blood withdrawn every 20 minutes for 24 hours, using a novel, discrete deconvolution algorithm to estimate apparent instantaneous secretory rates. The application of this methodology revealed that the frequency of discrete LH instantaneous secretory rates increased after mu opiate receptor blockade (P = 0.011). More strikingly, all parameters of testosterone secretory events responded significantly to mu opiate receptor blockade, including increases in mean estimated secretory rate (+47%, P = 0.02), testosterone pulse frequency (+ 64%, P less than 0.001) and amplitude (+ 20%, P = 0.027). Correspondingly, decreases in testosterone interpulse secretory intervals (-35%, P = 0.001), secretory pulse duration (-19%, P = 0.042) and interpulse valley duration (-35%, P = 0.006) also were noted. There was a prominent diurnal rhythm in testosterone secretion with maximal values in the morning and late evening, and marked reductions in the afternoon, sometimes to prepubertal levels. This variation in the testosterone secretory profile paralleled that of LH. In response to naltrexone, the FSH concentration series showed a significant increase in the mean FSH concentration (+ 18%) P = 0.003) and mean peak amplitude (+ 15%, P = 0.002). These data provide indirect evidence of functional coupling of the opiate system with the hypothalamic GnRH pulse generator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in the pulsatile properties of gonadotropin secretion in alcoholic men

The functional characteristics of the hypothalamic-pituitary-testicular axis were examined quantitatively in 10 chronic alcoholic men without hepatic dysfunction or clinical nutritional deficiencies. Spontaneous gonadotropin pulsatility was analyzed in blood sampled every 20 minutes over a 24-hour period 3 to 16 days after abstinence from alcohol and again 29 to 39 days later. The numbers of LH and FSH pulses per 24 hours were normal in these alcoholic men compared with controls. However, we found increased mean 24-hour concentrations of immunoactive LH (P = 0.012) and FSH (P = 0.018), increased peak heights for LH (P = 0.035) and FSH (P = 0.004), decreased fractional LH (P = 0.002) and FSH (P = 0.044) pulse amplitudes and increased interpulse valley mean LH (P = 0.010) and FSH (P = 0.018) concentrations. Serum levels of total testosterone, total estradiol and estrone were normal, whereas concentrations of free testosterone and free estradiol were increased. Pituitary release of LH and FSH was normal in response to low (5-micrograms) and high (95-micrograms) doses of GnRH given intravenously. The present observations indicate that in chronically alcoholic men, acute abstinence from ethanol is associated with elevated circulating concentrations of immunoactive gonadotropins in the presence of intact spontaneous gonadotropin pulsatility, preserved pituitary responsiveness to exogenous GnRH, and increased concentrations of free testosterone and free estradiol. Such findings are consistent with alterations in the endogenous feedback actions of sex steroid hormones in this setting.     

Primary gonadal failure in men selectively amplifies the mass of follicle stimulating hormone (FSH) secreted per burst and increases the disorderliness of FSH release patterns: reversibility with testosterone replacement

The neuroendocrine mechanisms by which primary gonadal failure in men increases mean serum FSH concentrations (castration-like response) are not known. To investigate the testosterone-dependent mechanisms of the FSH castration response: (i) blood was sampled at 10-min intervals for 24 h for later FSH assay in seven normal middle-aged men and in six patients with primary testicular failure, during testosterone withdrawal and after 6 weeks of parenteral testosterone replacement; (ii) using a specific two-site IRMA, serum FSH concentrations were measured, since this assay correlates well with an in-vitro Sertoli cell bioassay; (iii) multiparameter deconvolution analysis was then applied to estimate the frequency, amplitude, duration, and mass of underlying FSH secretory bursts, and the half-life of endogenous FSH, and (iv) approximate entropy was calculated to quantify the relative orderliness of FSH release over 24 h. Mean (+/- SEM) 24-h serum FSH concentrations were 3.9 +/- 0.8 IU/L in control subjects and 39 +/- 10 IU/L in unreplaced hypogonadal patients (p = 0.034). Deconvolution analysis revealed similar estimated mean FSH half-lives of 346 +/- 40 min (control) and 321 +/- 47 min (untreated patients), and indistinguishable FSH secretory burst frequencies, namely, 20 +/- 0.95 (normal) and 21 +/- 1.3 (patients) pulses per 24 h. In contrast, the daily production rate of FSH was markedly increased in testosterone-withdrawn hypogonadal men at 117 +/- 25 vs. 9.3 +/- 1.8 IU/L/day (control) (p < 0.01). This was due to a 10-fold higher calculated maximal rate (amplitude) of FSH secretion achieved within each FSH release episode (normal 0.078 +/- 0.02 vs. gonadal failure 0.74 +/- 0.087 IU/L/min, p < 0.01), yielding a 10-fold increase in the mass of FSH secreted per burst (control 0.53 +/- 0.06 vs. patients 5.3 +/- 0.81 IU/L, p < 0.01). In contrast, the mean half-duration of FSH secretory bursts was unaltered in unreplaced hypogonadal men at 8.2 +/- 2.2 min (control) vs. 7.0 +/- 1.0 min (patients). Approximate entropy (ApEn), a scale- and model-independent statistic designed to quantify the orderliness or regularity of hormone release, revealed greater irregularity of serum FSH concentrations in the hypoandrogenic state: ApEn = 1.8 +/- 0.025 (testosterone-withdrawn) vs. 1.6 +/- 0.037 (control) (p < 0.05). Parenteral testosterone replacement for 6 weeks significantly decreased mean serum FSH concentrations by reducing the daily FSH secretion rate and FSH secretory burst amplitude and mass, and concomitantly restored the orderliness of FSH release patterns. Testosterone treatment did not change FSH secretory burst half-duration, number, interburst interval, or half-life. It is concluded that primary gonadal failure in men evokes FSH hypersecretion which is marked by more disorderly FSH release patterns and a selectively amplified mass of FSH secreted per burst. These hypergonadotrophic mechanisms are, to a significant extent, testosterone-suppressible.     

24-hour pulsatile and circadian patterns of cortisol secretion in alcoholic men

Pulsatile and circadian patterns of cortisol secretion during acute (3 to 16 days) and chronic (29 to 39 days) abstinence were examined in alcoholic men with no clinical or laboratory evidence of hepatic dysfunction or nutritional deficiencies. Mean and integrated 24-hour serum concentrations of cortisol determined by sampling the blood every 20 minutes over a 24-hour period were increased in six out of 10 alcoholic subjects during acute abstinence when compared with normal controls. Sustained abstinence in seven subjects with follow-up studies caused significant decreases in the mean maximal cortisol peak amplitude (13 +/- 1.0 SEM acutely vs. 10.3 +/- 0.52 micrograms/dl follow-up; P = 0.01), mean 24-hour serum cortisol concentrations (10.9 micrograms/dl +/- 1.2 vs. 8.5 micrograms/dl +/- 0.26; P = 0.047), interpulse valley mean (9.3 micrograms/dl +/- 0.88 vs. 6.5 micrograms/dl +/- 0.34; P = 0.007), and valley nadir (7.9 micrograms/dl +/- 0.69 vs. 5.4 micrograms/dl +/- 0.30; P = 0.0036) concentrations. Cortisol pulse frequency was normal. Although circadian cortisol rhythmicity was maintained in alcoholics, the timing of the circadian acrophase was delayed significantly (P = 0.006) during acute abstinence (1022 [clocktime] +/- 34 min) as compared with normal controls (0743 [clocktime] +/- 34 min), and the amplitude of circadian cortisol rhythms exceeded normal in five of 10 alcoholics. Analysis of data in one alcoholic subject by a new multiparameter deconvolution method demonstrated increases in secretory burst amplitude (0.64 microgram/dl +/- 0.08 SD), mass of cortisol released per burst (9.8 micrograms/dl +/- 1.2 SD), and daily endogenous cortisol production rate (22 mg +/- 2.4 SD) during acute abstinence. These values were statistically different when compared with seven normal controls and the subjects' values during sustained abstinence (P less than 0.02). In conclusion, the results of the present study suggest increased daily production of cortisol as a possible mechanism underlying the elevated serum cortisol concentrations in chronic alcoholics during acute abstinence. This abnormality is shown to be reversible with sustained abstinence from alcohol.     

Kinetics of secretion of luteinizing hormone subunits and free alpha, and effects of gonadotropin releasing hormone

While GnRH is known to stimulate release of pituitary gonadotropins, its acute effects on the kinetics of secretion of the various hormones and subunits are not well characterized. Pulse-chase experiments were therefore performed to compare the time course of secretion of newly synthesized LH subunits and free alpha from rat pituitary quarters, and to study the effects of GnRH. After a 1-h pulse labeling with [35S]cysteine in the presence or absence of 10(-8) M GnRH, cultures were chased with excess unlabeled cysteine for 1, 2, 4, 8, or 20 h. Tissue lysates and media were immunoprecipitated sequentially with antisera to PRL, GH, LH beta and LH alpha, and the products were analyzed by gel electrophoresis. Labeled LH alpha was completely secreted by 4 h of chase without GnRH, and by 2 h with GnRH, as shown by its appearance in media and depletion from pituitary. Newly synthesized LH beta was depleted only at 8 h with GnRH from pituitary, suggesting much slower secretion. Incorporation of 35S into LH beta was approximately half that into LH alpha. Newly synthesized free alpha subunit was secreted between 4 and 20 h without GnRH, and by 8 h with GnRH. Free alpha incorporated a similar amount of radioactivity as LH beta. GnRH had no effect on the timing of the secretion of labeled total protein, PRL, GH, or TSH subunits. The amount of label incorporated during the pulse was greatest for PRL, approximately 15% of total protein label. GH incorporated approximately 20% as much label as PRL, with the glycoprotein subunits somewhat lower. The total amount of 35S incorporated into each protein studied was not affected by GnRH. The data indicate that short term in vitro GnRH exposure during a 1-h pulse labeling and chase acts to accelerate early secretion of LH alpha, LH beta and free alpha but does not affect overall protein synthesis. The peptide hormones PRL and GH incorporated the greatest amount of label and were the most briskly secreted hormones, while LH alpha led the glycoprotein subunits in the amount of incorporation and rapidity of secretion.     

Physiologic attributes of the luteinizing hormone pulse signal in the human. Cross-validation studies in men

The performance of two new statistically based, independently formulated, endocrine peak detection methodologies (Cluster and Detect) were tested on the same large set of physiologic LH pulsations. Serum LH concentrations were determined in blood samples withdrawn at 5-minute intervals for 24 hours in eight healthy young men. The subsequent comprehensive LH time series were subjected to Cluster and Detect analysis, each constrained to an alpha level of 0.01 or 0.05. Under both circumstances, computer estimates of physiologic LH pulse frequency by the two separate algorithms were statistically indistinguishable. Moreover, the two distinct methodologies were concordant not only in relation to the original 5-minute sampling series but also with respect to the constituent 10-, 15-, 20-, 30-, and 45-minute sampling data. The cross-validation of these two mathematically independent methodologies has permitted delineation of the detailed spectrum of spontaneous LH pulse properties within individual men and among different males under basal physiologic conditions.     

Pulsatile insulin secretion dictates systemic insulin delivery by regulating hepatic insulin extraction in humans

In health, insulin is secreted in discrete pulses into the portal vein, and the regulation of the rate of insulin secretion is accomplished by modulation of insulin pulse mass. Several lines of evidence suggest that the pattern of insulin delivery by the pancreas determines hepatic insulin clearance. In previous large animal studies, the amplitude of insulin pulses was related to the extent of insulin clearance. In humans (and in large animals), the amplitude of insulin oscillations is approximately 100-fold higher in the portal vein than in the systemic circulation, despite only a fivefold dilution, implying preferential hepatic extraction of insulin pulses. In the present study, by direct hepatic vein sampling in healthy humans, we sought to establish the extent of first-pass hepatic insulin extraction and to determine whether the pattern of insulin secretion (insulin pulse mass and amplitude) dictates the hepatic insulin clearance and thereby delivery of insulin to extrahepatic insulin-responsive tissues. Five nondiabetic subjects (two men and three women, mean age 32 years [range 25-39], BMI 24.9 kg/m(2) [21.2-27.1]) participated. Insulin and C-peptide delivery from the splanchnic bed was measured in basal overnight-fasted state and during a glucose infusion of 2 mg . kg(-1) . min(-1) by simultaneous sampling from the hepatic vein and an arterialized vein along with direct estimation of splanchnic blood flow. Fractional insulin extraction was calculated from the difference between the C-peptide and insulin delivery rates from the liver. The time patterns of insulin concentrations and hepatic insulin clearance were analyzed by deconvolution and Cluster analysis, respectively. Cross-correlation analysis was used to relate C-peptide secretion and insulin clearance. Glucose infusion increased peripheral glucose concentrations from 5.4 +/- 0.1 to 6.4 +/- 0.4 mmol/l (P < 0.05). Likewise, insulin and C-peptide concentrations increased during glucose infusion (P < 0.05). Hepatic insulin clearance increased with glucose infusion (1.06 +/- 0.18 vs. 2.55 +/- 0.38 pmol . kg(-1) . min(-1); P < 0.01), but fractional hepatic insulin clearance was stable (78.2 +/- 4.4 vs. 84 0. +/- 3.9%, respectively; P = 0.18). Insulin secretory-burst mass rose during glucose infusion (P < 0.05), whereas the interburst interval remained unchanged (4.4 +/- 0.2 vs. 4.5 +/- 0.3 min; P = 0.36). Cluster analysis identified an oscillatory pattern in insulin clearance, with peaks occurring approximately every 5 min. Cross-correlation analysis between prehepatic C-peptide secretion and hepatic insulin clearance demonstrated a significant positive association without detectable (<1 min) time lag. Insulin secretory-burst mass strongly predicted insulin clearance (r = 0.81, P = 0.0043). In conclusion, in humans, approximately 80% of insulin is extracted during the first liver passage. The liver rapidly responds to fluctuations in insulin secretion, preferentially extracting insulin delivered in pulses. The mass (and therefore amplitude) of insulin pulses traversing the liver is the predominant determinant of hepatic insulin clearance. Therefore, through this means, the pulse mass of insulin release dictates both hepatic (directly) as well as extra-hepatic (indirectly) insulin delivery. These findings emphasize the dual role of the liver and pancreas and their relationship mediated through magnitude of insulin pulse mass in regulating the quantity and pattern of systemic insulin delivery.     

Evidence that estrogen regulation of testosterone secretion in adult rams is mediated by both indirect (gonadotropin dependent) and direct (gonadotropin independent) means

Involvement of endogenous estrogen in the regulation of gonadotropin and testosterone secretion was investigated in adult rams. Groups of four rams were either passively immunized against estradiol or treated with the antiestrogen tamoxifen for 2 weeks during the breeding season (October). Circulating testosterone levels in immunized rams increased eight-fold to supraphysiologic values as episodic elevations and baseline levels increased in magnitude; only moderate increases in LH peak frequency and magnitude occurred, and prolactin fell to undetectable levels. Tamoxifen treatment was not associated with changes in mean hormone levels, although there was a tendency toward reductions in the magnitude of episodic LH and testosterone secretion. When rams were challenged with exogenous GnRH and LH, a greater testicular endocrine response was observed in the immunized rams and the pituitary endocrine response was delayed in the tamoxifen-treated rams. Results indicate that in the ram 1) circulating levels of estradiol provide negative feedback signals of different intensities to the testis and the hypothalamic-pituitary axis and 2) tamoxifen exerts a mild estrogenic effect when administered at the dose of 25 mg/day.     

Negative feedback regulation of pulsatile LH secretion during treatment with an LHRH antagonist in rams

Suppression of LH and testosterone secretion in sexually active rams by the short-term administration of an LHRH antagonist results in a compensatory increase in the release of LHRH from the hypothalamus. This is inferred from the observed increase in the frequency of LH pulses in peripheral blood during the period of recovery when the pituitary regains its responsiveness to LHRH. To investigate the nature of the inhibitory feedback signal which triggers this compensatory response, a single intravenous injection of 1 mg of an LHRH antagonist (28 micrograms/kg; N-Ac-D-pCl-Phe 1, D-pCl-Phe 2, D-Trp 3, D-hArg (Et 2) 6, D-Ala 10, LHRH) was given to groups of intact, testosterone-implanted castrated and castrated rams housed under stimulatory short days. Pulsatile LH secretion was monitored in blood samples collected every 10 min for 34 h. The treatment caused an immediate blockade of LH pulses in all three groups of rams followed by a progressive recovery of LH secretion from 12-30 h. Compared to the pretreatment period, intact rams showed a significant increase in frequency of LH pulses during the recovery period. Castrated rams did not show this increase, with or without supplementary testosterone. Since the circulating testosterone concentration decreased after the blockage of LH secretion in the intact rams, but not in the castrated or testosterone-implanted castrated rams, we conclude that it is the reduction in the steroid negative feedback signal which leads to a compensatory increase in the activity of the LH pulse generator.     

Differential regulation in the release of bioactive versus immunoactive gonadotropins from cultured rat pituitary cells by inhibin and androgens.

Using in vitro bioassays and radioimmunoassays, the authors examined the interactions between purified 32kD ovine inhibin, testosterone, and dihydrotestosterone on basal and GnRH-stimulated secretion of gonadotropins in primary rat pituitary cell cultures. This study demonstrates that inhibin and androgens: 1) differentially regulate gonadotropin levels, 2) cause changes in the amounts of bioactive and immunoactive FSH and LH released. In conclusion, differences in bio- and immuno-FSH and LH secretion suggest that the endocrine milieu results in not only quantitative but qualitative changes in the secreted gonadotropin isoforms.     

Pulsatile secretion of gonadotropins and prolactin in male marathon runners. Relation to the endogenous opiate system

We tested the hypothesis that sustained, strenuous physical training alters the neuroendocrine regulation of pulsatile gonadotropin and/or prolactin secretion in men. Blood was sampled at 20-minute intervals over 8 hours in five endurance-trained men after a 10-15 mile run in the middle of the active training season, and in 11 nonendurance trained normal controls. In these two groups, basal patterns of physiologically pulsatile secretion of LH, FSH, and prolactin (PRL) were not significantly different in relation to the following parameters: mean serum concentration of each of the three hormones (N = 25 samples); areas under the hormone concentration vs. time curves; fractional, incremental, and absolute pulse amplitudes; and pulse frequency, or periodicity. To test for enhanced suppressive effects of endogenous opiates in trained male marathon runners, subjects were administered the potent opiate-receptor antagonist, naltrexone (1 mg/kg). This antagonist significantly stimulated pulsatile LH secretion by increasing mean serum LH values from 10.94 to 13.58 mIU/ml (P = 0.007); area under the LH concentration vs. time curve increased from 5370 to 6510 mIU/ml X 8 hours (P = 0.05) and, pulse frequency rose from 2.8 to 4.9 pulses/8 hours (P = 0.006). Naltrexone also enhanced pulse frequency of FSH secretion from 3.4 to 5.4 pulses/8 hours (P = 0.009), but did not alter serum prolactin concentrations. None of these responses differed significantly from those in normal sedentary controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential patterns of inhibin secretion in response to gonadotrophin stimulation in normal men

Inhibin B is produced by the testis, and its constituent alpha and beta B subunits have been localized immunohistochemically to Leydig as well as Sertoli cells in both rodent and human testes. Whether Leydig cells contribute to circulating inhibin B concentrations, however, is uncertain. We have investigated this by selectively stimulating Leydig and Sertoli cells with hCG and FSH, respectively. The study was a randomized crossover trial, investigating responses to 225 IU recombinant FSH or 3000 IU hCG administered s/c 4-6 weeks apart. Ten normal men were recruited to participate. Blood was taken twice before treatment and after 8, 24, 48, 72 and 96 h. Serum was assayed for FSH, LH and testosterone by radioimmunoassay (RIA); inhibin B and pro-alpha C inhibin forms by ELISA. Administration of hCG, but not FSH, caused a rapid increase in blood testosterone levels, which reached a maximum after 72 h (22.2 +/- 2.7-50.1 +/- 4.5 nmol/L, p < 0.001). Inhibin B concentrations in blood were unchanged following either treatment. Conversely, pro-alpha C concentrations increased following both treatments. FSH administration resulted in a gradual increase in pro-alpha C concentrations (369 +/- 18 pg/mL pre-treatment to 453 +/- 33 pg/mL after 96 h, p=0.013). Administration of hCG resulted in a more rapid response, with pro-alpha C concentrations rising from 384 +/- 23 pg/mL pre-treatment to a peak at 48 h of 535 +/- 45 pg/mL (p=0.007). This response was more rapid than that of testosterone. These results demonstrate that adult human Leydig, as well as Sertoli, cells secrete inhibin alpha subunit in response to gonadotrophin stimulation but provide no evidence for the secretion of inhibin B from Leydig cells. The lack of change in inhibin B secretion in response to FSH suggests that more prolonged or intense stimulation of Sertoli cells may be required for secretion of the dimeric form.     

Effect of modulating endogenous prolactin secretion on testosterone production in the adult male bonnet monkey (Macaca radiata):

Adult male bonnet monkeys maintained under regulated light: dark conditions exhibit a nycthemeral surge of testosterone. The present study attempts to determine the effect of administration of drugs that modulate prolactin levels like ergobromocriptine (EBC) and chlorpromazine (CPZ) on testosterone production. The injection of EBC, a known inhibitor of prolactin secretion, could abolish nocturnal testosterone surge irrespective of the drug being given at 8.00 or 17.00 h. Testosterone surge could likewise be inhibited by treating animals with CPZ, a potent stimulator of prolactin secretion. This suggests that alteration in endogenous prolactin level from the normal effects nycthemeral surges of testosterone. The in vivo responsiveness of the testes of monkeys injected either CPZ oder EBC to exogenous LH or LHRH stimulation was tested. While LH could completely override the CPZ induced inhibition in testosterone production it could only partially reverse the EBC effect.