Liu, Peter Y.

The long-lived glycoprotein hormone, human chorionic gonadotropin (hCG), downregulates testosterone (T) biosynthesis in vitro and in vivo in animals and humans. The degree to which short-lived pulses of pituitary luteinizing hormone (LH) do so, particularly at physiological concentrations, is not known. We test the hypothesis that continuous LH infusion compared with bolus injections of LH every 1 h or every 2 h overnight downregulates T secretory responses to a subsequent fixed template of three consecutive intravenous pulses of a physiological amount of recombinant human (rh) LH (triple stimulus). Nineteen healthy men ages 18–49 yr each underwent four separate randomly ordered overnight gonadotropin-releasing hormone-receptor antagonist treatments with superimposed intravenous infusions of saline or rhLH (1-h pulses, 2-h pulses, or continuously). Each 12-h infusion protocol was followed by the triple rhLH-pulse stimulus the next morning. During the triple stimulus, basal (nonpulsatile) as well as total (basal plus pulsatile) T secretion was higher after overnight 2- and 1-h rhLH pulses than after continuous rhLH or saline delivery. Approximate entropy, a probabilistic measure of feedforward-induced irregularity of T concentration time series, was higher after 1-h rhLH pulses than after continuous rhLH. Analytical estimation of pulsatile rhLH-T dose-response measures revealed higher T secretory sensitivity and greater rhLH potency (lower EC50) after exposure to 1-h than 2-h rhLH pulses. Collectively, these data indicate that in vivo dynamics of LH-stimulated T secretion under standardized conditions in men depend on the prior time mode of LH delivery in the bloodstream.

Direct sampling of the human spermatic veins has disclosed concomitant LH and testosterone (T) pulses, suggesting pulsatile LH concentration-dependent stimulation of T secretion. However, studies to date have examined this hypothesis using only pharmacological stimulation with hCG. The present study tests the hypothesis that age is marked by decreased T secretory responses to repeated near-physiological iv pulses of recombinant human LH administered in a Clinical Translational Science Center. Participants included 92 healthy men aged 18–75 yr with BMI 18–34 kg/m2. The contribution of endogenous LH pulses was minimized by combined injection of a selective GnRH receptor antagonist sc and successive pulses of biosynthetic LH iv. A new analytical dose response model was applied to estimate the properties of exogenous LH's drive of T secretion. Regression of LH-T dose response potency estimates on age showed that the efficacy of pulses of biosynthetic LH progressively decreased with age ( P = 0.014, r = 0.26). Testis sensitivity to exogenous LH pulses also declined with age ( P = 0.011, r = 0.27). Moreover, estimated Leydig cell downregulation by LH pulses rose significantly with age ( P = 0.039, r = 0.22). These outcomes were selective, since the recovery potency of infused LH was not affected by age but was reduced by increasing BMI ( P = 0.011, r = 0.27). Assuming stable bioactivity of infused recombinant human LH, these novel data indicate that factors associated with age and BMI attenuate LH efficacy and testis sensitivity and augment Leydig cell downregulation in healthy men.

Negative-feedback (inhibitory) and positive-feedforward (stimulatory) processes regulate physiological systems. Whether such processes are themselves rhythmic is not known. Here, we apply cross-approximate entropy (cross-ApEn), a noninvasive measurement of joint (pairwise) signal synchrony, to inferentially assess hypothesized circadian and ultradian variations in feedback coupling. The data comprised simultaneous measurements of three pituitary and one peripheral hormone (LH, FSH, prolactin, and testosterone) in 12 healthy men each sampled every 10 min for 4 days (5,760 min). Ergodicity, due to the time series stationarity of the measurements over the 4 days, allows for effective estimation of parameters based upon the 12 subjects. Cross-ApEn changes were quantified via moving-window estimates applied to 4-day time series pairs. The resultant ordered windowed cross-ApEn series (in time) were subjected to power spectrum analysis. Rhythmicity was assessed against the null hypothesis of randomness using 1,000 simulated periodograms derived by shuffling the interpulse-interval hormone-concentration segments and redoing cross-ApEn windows and spectral analysis. By forward cross-ApEn analysis, paired LH-testosterone, LH-prolactin, and LH-FSH synchrony maintained dominant rhythms with periodicities of 18–22.5, 18, and 22.5 h, respectively (each P < 0.001). By reverse (feedback) cross-ApEn analysis, testosterone-LH, testosterone-prolactin, and testosterone-FSH synchrony cycles were 30, 18, and 30–45 h, respectively (each P ≤ 0.001). Significant 8- or 24-h rhythms were also detected in most linkages, and maximal bihormonal synchrony occurred consistently at ∼0400–0500. Collectively, these analyses demonstrate significant ultradian (<24 h), circadian (∼24 h), and infradian (>24 h) oscillations in pituitary-testis synchrony, wherein maximal biglandular coordination is strongly constrained to the early morning hours.

Testosterone (T) exerts negative feedback on the hypothalamo-pituitary (GnRH-LH) unit, but the relative roles of the CNS and pituitary are not established. We postulated that relatively greater LH responses to flutamide (brain-permeant antiandrogen) than bicalutamide (brain-impermeant antiandrogen) should reflect greater feedback via CNS than pituitary/peripheral androgen receptor-dependent pathways. To this end, 24 healthy men ages 20–73 yr, BMI 21–32 kg/m2, participated in a prospective, placebo-controlled, randomized, double-blind crossover study of the effects of antiandrogen control of pulsatile, basal, and entropic (pattern regularity) measurements of LH secretion. Analysis of covariance revealed that flutamide but not bicalutamide 1) increased pulsatile LH secretion ( P = 0.003), 2) potentiated the age-related abbreviation of LH secretory bursts ( P = 0.025), 3) suppressed incremental GnRH-induced LH release ( P = 0.015), and 4) decreased the regularity of GnRH-stimulated LH release ( P = 0.012). Furthermore, the effect of flutamide exceeded that of bicalutamide in 1) raising mean LH ( P = 0.002) and T ( P = 0.017) concentrations, 2) accelerating LH pulse frequency ( P = 0.013), 3) amplifying total (basal plus pulsatile) LH ( P = 0.002) and T ( P < 0.001) secretion, 4) shortening LH secretory bursts ( P = 0.032), and 5) reducing LH secretory regularity ( P < 0.001). Both flutamide and bicalutamide elevated basal (nonpulsatile) LH secretion ( P < 0.001). These data suggest the hypothesis that topographically selective androgen receptor pathways mediate brain-predominant and pituitary-dependent feedback mechanisms in healthy men.

Abstract Background: Healthy older men have reduced testosterone (Te) production and frequent, small irregular LH pulses. Which is cause and which is effect are not known. Rationale: In model systems, frequent and irregular LH pulses reflect attenuated feedback inhibition by Te. Hypothesis: Factors associated with aging impair negative feedback by Te. Subjects and Setting: Healthy men at an academic medical center were studied. Methods: The study used quantification of the regularity of LH release patterns during blockade of gonadal steroidogenesis and graded transdermal Te addback in 18 healthy men aged 18–65 yr. Results: In the cohort as a whole, stepwise Te repletion repressed LH concentrations (P = 0.001) and enhanced the quantifiable orderliness of LH release patterns (P &lt; 0.001). By regression analysis, age attenuated the capability of increasing Te concentrations to regularize LH secretion patterns (P = 0.019). However, after a fixed GnRH stimulus, the effect of Te on LH regularity was invariant of the age factor (P = 0.16), thus pointing to a hypothalamic locus of impaired Te feedback. Summary: Aging disrupts the capability of systemic Te concentrations to maintain orderly LH secretion under endogenous, but not exogenous, GnRH drive. Conclusions: Age or factors associated with increased age reduce negative feedback by any given total Te concentration on hypothalamopituitary GnRH-LH outflow, thus contributing to disorderly LH secretion.

Abstract Background: Controversy exists over the association of estrogen and cardiovascular disease. Estrogen receptors (ERs) α and β are expressed in the endothelial cells and vascular smooth muscle cells (VSMCs) of many arteries, but the relative importance of ERα or ERβ in mediating the vascular response to estrogens is not well defined, particularly in humans. We have shown previously that postmenopausal women receiving hormone therapy (HT) had lower mean coronary artery calcium, plaque area, and calcium-to-plaque ratio compared with untreated women. In this study, we examined coronary artery ERα and ERβ expression in pre- and postmenopausal women as a function of plaque area, calcium area, calcium-to-plaque ratio, and estrogen status. Methods: Coronary arteries were obtained at autopsy from a total of 55 women: nine premenopausal women, 13 postmenopausal women on HT and 33 untreated postmenopausal women (non-HT). Coronary calcification was quantified by contact microradiography, and atherosclerotic plaque area was measured histologically. Coronary artery cross-sections were immunostained for ERα and ERβ, and the amount of receptors was estimated semiquantitatively in each arterial wall layer (intima, adventitia, and media). Double immunofluorescence was used to colocalize ERα and ERβ with smooth muscle actin, a marker of VSMCs. Results: ERβ and ERα were expressed in all artery wall layers, but most avidly in the media (P = 0.001), and colocalized with VSMCs. ERβ expression exceeded ERα expression in all wall layers (P &lt; 0.001) and was adjacent to areas of calcium deposition. ERβ expression in the intimal layer correlated with calcium content, plaque area, and calcium-to-plaque ratio (all P &lt; 0.01) and tended to be greater in non-HT than in HT women (P = 0.06). ERα expression did not vary significantly among groups, nor did it correlate with calcium content, plaque area or calcium-to-plaque ratio. Expression of ERα but not ERβ declined with age (P &lt; 0.01) in HT women only. Age had no effect on ERα or ERβ expression in non-HT or premenopausal women. Conclusions: ERβ is the predominant ER in human coronary arteries and correlates with coronary calcification, a marker of severe atherosclerosis. Increased ERβ expression is linked to advanced atherosclerosis and calcification independent of age or hormone status. Future pharmacogenetic studies that target this receptor are needed to confirm causality.

Context: Testosterone (Te) depletion in aging men in principle could reflect deficits in the hypothalamus, pituitary gland, or testis. Available pharmacological studies of possible failure of Leydig cell steroidogenesis remain inconclusive. Objective: The objective of the study was to assess Te secretion in older and young men in response to near physiological LH stimulation. Intervention: Pulsatile iv infusion of recombinant human LH was administered for 2 d to stimulate Te secretion during suppression of endogenous LH concentrations with a potent selective GnRH receptor antagonist (ganirelix). Subjects/Context: Healthy older (aged 60–73 yr, n = 8) and young (19–30 yr, n = 13) men were studied in an academic setting. Measures: Pulsatile LH and Te concentrations on the second day of exogenous LH stimulation were measured. Results: Serum ganirelix concentrations and infused LH pulse increments were similar by age. In contrast, older subjects manifested: 1) reduced mean Te concentrations (P = 0.016), Te peak heights (P = 0.014), increments (P = 0.010), summed areas (P &lt; 0.013), and interpeak Te concentrations (P = 0.023); 2) decreased Te to LH concentration ratios (P = 0.002); 3) diminished LH-Te feed-forward synchrony (P = 0.020); and 4) a blunted amplitude (P = 0.036) and advanced phase (P = 0.013) of diurnal Te rhythms. Conclusion: A novel regimen of pulsatile LH stimulation for 48 h during GnRH receptor blockade unmasks deficits in pulsatile, basal, synchronous, and nyctohemeral Te secretion in healthy older men. These findings do not exclude concomitant defects in GnRH outflow and/or Te-negative feedback in the aging male.

Healthy older men manifest concomitant hypoandrogenemia and attenuation of LH pulse size. Because exogenous GnRH remains effective, a plausible intuition is that aging reduces hypothalamic GnRH secretion, thus mediating relative hypogonadotropic hypogonadism. To assess the impact of age on central GnRH outflow indirectly, we quantitated graded suppression of pulsatile LH secretion by saline and escalating doses of a potent and selective GnRH-receptor antagonist, ganirelix, in 18 healthy men ages 23–72 yr. The rationale is that ganirelix should reduce the amplitude of LH pulses in proportion to both drug concentration and endogenous GnRH feedforward. To this end, blood was sampled every 10 min for 2 h before and 16 h after sc administration of saline or ganirelix and for 3 additional hours after iv injection of a fixed dose of GnRH (100 ng/kg); concentrations of LH and ganirelix were measured by immunochemiluminometry and RIA, respectively; and pulsatile LH secretion was quantitated by a deconvolution procedure. Log-linear regression analysis was used to estimate the sensitivity of pulsatile LH secretion to inhibition by a unit increase in serum ganirelix concentrations in each subject. Statistical analyses revealed that increasing age markedly attenuated the capability of ganirelix to decrease LH pulse size (viz., r = −0.648; P = 0.004). In contrast, age did not modify the competitive interaction between injected GnRH and ganirelix. These joint outcomes support the clinical hypothesis that age diminishes hypothalamic GnRH outflow without impairing GnRH action in healthy men.