In men, peripheral estradiol levels directly reflect the action of estrogens at the hypothalamo-pituitary level to inhibit gonadotropin secretion

CONTEXT: Estradiol inhibits gonadotropin release in men by an action at the hypothalamus and pituitary. Because of the tissue-specific regulation of aromatase, peripheral estradiol levels may not reflect brain estradiol concentrations. OBJECTIVE: We evaluated whether local aromatization of testosterone in the hypothalamus or pituitary is important for gonadotropin release and to what extent circulating estrogens affect gonadotropin levels and peripheral testosterone levels. DESIGN, SUBJECTS, AND INTERVENTIONS: We suppressed aromatase activity in 10 young healthy men with letrozole 2.5 mg once daily, restored plasma estradiol levels with estradiol patches (100 microg/d for the first week, 50 microg/d the second week, 25 microg/d the third week, and no estradiol patch the fourth week) and measured plasma testosterone, estradiol, LH, FSH, and SHBG levels. RESULTS: The mean estradiol and testosterone levels during the study ranged between 68.6 +/- 38.3 and 12.6 +/- 7.21 pg/ml for estradiol and 179 +/- 91 and 955 +/- 292 ng/dl (mean +/- sd) for testosterone. Levels of testosterone, LH, and FSH were inversely related to peripheral estradiol levels. During letrozole use, the mean plasma estradiol level needed to restore testosterone, LH, and FSH levels to baseline levels was not significantly different from the baseline mean estradiol level. CONCLUSIONS: Local aromatization of testosterone in the hypothalamo-pituitary compartment is not a prerequisite for expression of the inhibitory action of estrogens on gonadotropin secretion in men. Peripheral estradiol levels directly reflect the inhibitory tone exerted by estrogens on gonadotropin release and are a major determinant of peripheral testosterone, LH, and FSH levels.     

Oestradiol replacement treatment and glucose homeostasis in two men with congenital aromatase deficiency: evidence for a role of oestradiol and sex steroids imbalance on insulin sensitivity in men.

AIMS: The role of sex steroids in glucose and insulin metabolism in men remains unclear. To investigate the effects of sex steroids and oestrogen on insulin sensitivity in men, we studied two male adults with aromatase deficiency (subject 1 and subject 2). METHODS: The effects of transdermal oestradiol (tE(2)) treatment at different dosages on insulin sensitivity were studied before tE(2) treatment (phase 1), and after 6 months (phase 2) and 12 months of tE(2) treatment (phase 3) by means of homeostasis model assessment-insulin resistance (HOMA-IR) and Quantitative Insulin Sensitivity Check Index (QUICKI), insulin tolerance test (ITT), and oral glucose tolerance test (OGTT). The latter was performed only in subject 1, as subject 2 suffered from Type 2 diabetes. RESULTS: The restoration of normal serum oestradiol led to improved insulin sensitivity, as shown by changes in HOMA-IR and QUICKI. The ITT provided evidence of improved insulin sensitivity during tE(2) treatment. Insulin secretion after OGTT was reduced during tE(2) treatment in subject 1. After 12 months of tE(2) treatment, insulin sensitivity was improved compared with in phases 1 and 2. CONCLUSIONS: The study suggests a direct involvement of oestrogens in insulin sensitivity, and supports a possible role of oestradiol : testosterone ratio, which may be as influencial as the separate actions of each sex steroid on glucose homeostasis.     

Activation of the somatotropic axis by testosterone in adult men: evidence for a role of hypothalamic growth hormone-releasing hormone

Testosterone (T) is known to affect the growth hormone (GH) axis. However, the mechanisms underlying the activation of GH secretion by T still remain to be clarified. Available data in animals and humans have shown that withdrawal of somatostatin (SRIH) infusion induces a GH-releasing hormone (GHRH)-mediated rebound release of GH, and there is accumulating evidence that SRIH infusion withdrawal may be a useful test to probe the GHRH function in vivo. With the aim of investigating whether the stimulatory effect of androgens on GH release in man could be accounted for by activation of the hypothalamic GHRH tone, we evaluated the plasma GH response to SRIH withdrawal in 10 patients aged 29.6 +/- 2.4 years (mean +/- SEM), diagnosed with hypergonadotropic hypogonadism, before and after a 6-month replacement therapy with T enanthate (250 mg every 3 weeks, i.m.), and in 10 healthy men, aged 26.7 +/- 2.8 years. To verify whether the modulation of GH secretion by T could also be mediated through changes in SRIH tone and/or pituitary releasable pool, we examined GH secretory responses to combined GHRH and L-arginine (ARG) in the same individuals. Basal plasma concentrations of GH (0.48 +/- 0.11 microg/l) and IGF-I (23.79 +/- 1.83 nmol/l) were significantly lower in untreated hypogonadal patients than in healthy men, and significantly increased after T replacement therapy (GH 1.13 +/- 0.28 microg/l; IGF-I 28.71 +/- 1.46 nmol/l). The mean Delta GH peak after SRIH withdrawal recorded in untreated hypogonadal men (2.65 +/- 0.86 microg/l) was significantly (p < 0.05) lower than that observed in healthy men (6.53 +/- 1.33 microg/l) and significantly increased after T replacement therapy (5.52 +/- 1.25 microg/l). The GH responses to GHRH combined with ARG (a functional SRIH antagonist) were not significantly different between healthy men and untreated hypogonadal patients, and were not significantly affected by T treatment. Plasma T and estradiol (E(2)) levels significantly correlated with Delta GH peak after SRIH withdrawal in healthy men and in T-treated hypogonadal patients, whereas in untreated patients they did not. No significant correlation was found between GH areas under the curve after GHRH + ARG test and T and E(2) plasma levels in either healthy men or in hypogonadal patients (both before and after T replacement). These findings are consistent with the view that in humans the stimulatory action of T on the GH axis appears to be mediated at the hypothalamic level primarily by promoting GHRH function.     

Pulsatile gonadotropin secretion in women with hypothalamic amenorrhea: evidence that reduced frequency of gonadotropin-releasing hormone secretion is the mechanism of persistent anovulation

Hypothalamic amenorrhea (HA) is a clinical disorder of unknown etiology. The diagnosis is made by exclusion of known abnormalities of pituitary and ovarian function. To determine if abnormalities of GnRH secretion could account for the anovulation and amenorrhea, we measured plasma gonadotropins every 20 min for 10- to 24-h periods in 19 women with HA. Ovarian steroids and gonadotropin responses to an iv bolus dose of GnRH (25 ng/kg) were also measured. The results were compared to those obtained during the early follicular (EF) and late luteal (LL) phases of ovulatory cycles in normal women. Plasma estradiol was lower (mean +/- SE, 52 +/- 5 pg/ml) than either cycle stage in normal women. Mean plasma LH was lower than EF values and FSH was higher than LL values. The amplitude of LH pulses in HA was similar to that in normal women. LH pulse frequency was the same as that present during the LL, but lower than that during the EF (HA, 4.7 pulses/12 h; EF, 7.7 pulses/12 h; P less than 0.05). In addition to the similar frequency, the patterns of LH secretion in HA resembled that of LL in that the amplitude of LH pulses was highly variable and pulses occurred at irregular intervals. Consistent changes in diurnal gonadotropin secretion were not found, and LH secretion was greater at night in 9 studies and during the day in 5 studies. Repeat studies in three patients (5-13 months later) revealed that LH pulse frequency was variable, being unchanged in 1, increased in 1, and decreased in the third patient. Thus, LH pulse frequency and, by inference, GnRH pulse frequency are similar in HA to those in the normal luteal phase despite a different steroid milieu. GnRH pulse frequency increases from the luteal to the follicular phases of normal cycles and may be important in the initiation of ovarian follicular maturation. These data suggest that the absence of cyclical gonadotropin secretion and anovulation in HA result from a decreased frequency and irregular amplitude of GnRH secretion and consequent absence of ovarian follicular maturation.     

Influence of diabetes on the gonadotropin response to the negative feedback effect of testosterone and hypothalamic neurotransmitter turnover in adult male rats.

The influence of diabetes on the gonadotropin response to the negative feedback effect of testosterone (T) and hypothalamic neurotransmitter turnover rates in adult male rats was evaluated. Adult male Sprague-Dawley rats were made diabetic by an intraperitoneal injection of streptozotocin (STZ; 5 mg/100 g body weight) in citrate buffer. Vehicle-injected rats served as controls. On day 9, all rats were bilaterally castrated and treated subcutaneously on alternate days with either peanut oil or T propionate (TP) in peanut oil (100 micrograms/rat). Plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin (PRL), and T concentrations were measured by specific radioimmunoassays from blood samples collected on day 1 (before castration) and 2, 4, 6, and 7 days after castration. On day 7 after castration (day 15 after vehicle or STZ treatment), 1 h before autopsy, the rats were injected intraperitoneally with saline or a tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine (25 mg/100 g BW), for the measurement of norepinephrine (NE) and dopamine turnover in median eminence and medial basal hypothalamus (MBH). Circulating FSH, LH, PRL, and T levels were significantly lower (FSH and T: p less than 0.001; LH and PRL: p less than 0.05) in gonad-intact rats treated with STZ than in vehicle-injected animals. The castration-induced increase in plasma LH levels was attenuated in diabetic rats. The suppressive effect of T on LH secretion was significantly greater (p less than 0.001) in STZ-treated rats relative to TP-treated nondiabetic controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indirect evidence for short-loop negative feedback of insulin secretion in the rat

Feedback inhibition of glucose-mediated insulin release has repeatedly been demonstrated in isolated pancreatic islets and in the perfused pancreas. It was the aim of the present study to determine whether inhibition occurs through a long-loop (plasma concentration of insulin) or a short-loop (local concentration) action of insulin. The perfused rat pancreas was used, with different perfusion rates and different insulin concentrations in the medium. Increasing the flow rate from 1 to either 3 or 6 ml/min gradually decreased the insulin concentration in the effluent, at stimulatory concentrations of glucose (11.1 and 16.7 mmol/l). Under the same conditions, however, the integrated amount of insulin released over a period of 30 min was significantly enhanced. When exogenous insulin (2.7 and 5.4 mumol/l) was added to the perfusion medium, insulin secretion in the presence of 11.1 or 16.7 mmol glucose/l at flow rates of 3 and 6 ml/min was diminished. This effect was most prominent with 11.1 mmol glucose/l and 2.7 mumol exogenous insulin/l at all flow rates (except 1 ml/min), as well as at the high perfusion flow rates with other glucose concentrations. Insulin secretion was not affected by 5.4 mumol exogenous insulin/l at 1 ml/min or by 2.7 mumol exogenous insulin/l at 3 ml/min. The data support a negative feedback inhibition of insulin secretion by secreted insulin, since insulin secretion was decreased by either adding exogenous insulin or by lowering endogenous insulin as the consequence of increased flow rates.(ABSTRACT TRUNCATED AT 250 WORDS)     

The negative feedback effects of testicular steroids are predominantly at the hypothalamus in the ram

This study aimed to delineate the hypothalamic and/or pituitary actions of testosterone and its primary metabolites 5 alpha-dihydrotestosterone and estradiol (E) in adult castrated rams (wethers) during the breeding season. In Exp 1, wethers were treated for a week with twice daily injections (im) of peanut oil, 8, 16 or 32 mg/day testosterone propionate (TP) or dihydrotestosterone benzoate (DHTB) or an sc silastic implant containing 1 or 3 cm E. TP decreased plasma LH concentrations, increased (P less than 0.05) LH interpulse interval, did not have consistent effects on LH pulse amplitude, and had minimal effects on plasma FSH concentrations. DHTB decreased LH and FSH concentrations and increased (P less than 0.05) LH interpulse interval. E reduced (P less than 0.05) plasma LH and FSH concentrations and increased LH interpulse interval but had no effects on LH pulse amplitude. In Exp 2, hypothalamo-pituitary disconnected wethers given 125 ng GnRH every 2 h, were treated with either peanut oil, 32 mg/day TP or DHTB or 3 cm E. None of the treatments affected plasma LH or FSH concentrations or LH pulse amplitude. Exp 3 investigated the effects on GnRH of treatment of wethers either with peanut oil or TP. TP reduced GnRH concentrations (P less than 0.05) and pulse amplitude (P less than 0.01) and increased interpulse interval (P less than 0.05). These data provide evidence that, during the breeding season, the principal site of negative feedback of testicular steroids in the ram is the hypothalamus, resulting in decreased GnRH secretion; feedback effects at the pituitary are minimal.     

Chronic human chorionic gonadotropin administration in normal men: evidence that follicle-stimulating hormone is necessary for the maintenance of quantitatively normal spermatogenesis in man

The role of FSH in the maintenance of spermatogenesis in man is poorly understood. To determine whether normal serum levels of FSH are necessary for the maintenance of quantitatively normal spermatogenesis, we first studied the effect on sperm production of selective FSH deficiency induced by chronic administration of hCG in normal men. Then, we determined the effect of FSH replacement in some of these men. After a 3-month control period, eight normal men (aged 30-39 yr) received 5000 IU hCG, im, twice weekly for 7 months. Then while continuing the same dosage of hCG, subjects simultaneously received 200 mg testosterone enanthate (T), im, weekly for an additional 6 months. hCG administration alone resulted in partial suppression of the mean sperm concentration from 88 +/- 24 (+/-SEM) million/ml during the control period to 22 +/- 7 million/ml during the last 4 months of hCG treatment (P less than 0.001 compared to control values). With the addition of T to hCG, sperm counts remained suppressed to the same degree. Except for one man who became azoospermic while receiving hCG plus T, sperm motilities and morphologies remained normal in all subjects throughout the entire study. During both the hCG alone and hCG plus T periods, serum FSH levels were undetectable (less than 25 ng/ml), and urinary FSH levels were comparable to those in prepubertal children and hypogonadotropic hypogonadal adults. We replaced FSH activity in four of the eight men in whom prolonged selective FSH deficiency and partial suppression of sperm production were induced by hCG administration. Immediately after the period of hCG plus T administration, T was stopped in four men who continued to receive hCG alone (5000 IU, im, twice weekly) for 3 months. Then, while continuing the same dosage of hCG, these men received 100 IU human FSH, sc, daily (n = 2) or 75 IU human menopausal gonadotropin, sc, daily (n = 2) for 5-8 months. During the second period of hCG administration alone, serum FSH levels were undetectable (less than 25 ng/ml), and sperm concentrations were suppressed (34 +/- 13 million/ml) compared to the control values for these four men (125 +/- 39 million/ml; P less than 0.001). With the addition of FSH to hCG, FSH levels increased (213 +/- 72 ng/ml) and sperm concentrations rose significantly, reaching a mean of 103 +/- 30 million/ml (P less than 0.03 compared to hCG alone).(ABSTRACT TRUNCATED AT 400 WORDS)     

Fast axonal transport in the presence of high Ca2+: evidence that microtubules are not required.

Microtubules have long been associated with the mechanism of fast axoplasmic transport, although experimental evidence to support an involvement has been equivocal. Electron microscopic studies demonstrated that incubation of the axons of excised rat sciatic nerves in media containing 75 mM Ca2+ caused complete loss of microtubules within 6 hr. To evaluate the role of microtubules in fast anterograde transport, studies of transport in nerves exposed to these conditions were undertaken. Prior to measurement of axoplasmic transport, nerves ligated distal to the dorsal root ganglia were preincubated in vitro in 75 mM Ca2+ for 0-6 hr. Fast axonal transport was subsequently monitored by measuring the amount of trichloroacetic acid-insoluble radioactivity that accumulated at the ligature after incubation for 12-18 hr with L-[3H]proline. Nerves in which microtubules had been depolymerized by preincubation in high Ca2+ maintained control levels of transport. We conclude that intact microtubules are not required for fast anterograde axoplasmic transport.     

A Fall in plasma free fatty acid (FFA) level activates the hypothalamic-pituitary-adrenal axis independent of plasma glucose: evidence for brain sensing of circulating FFA

The brain responds to a fall in blood glucose by activating neuroendocrine mechanisms for its restoration. It is unclear whether the brain also responds to a fall in plasma free fatty acids (FFA) to activate mechanisms for its restoration. We examined whether lowering plasma FFA increases plasma corticosterone or catecholamine levels and, if so, whether the brain is involved in these responses. Plasma FFA levels were lowered in rats with three independent antilipolytic agents: nicotinic acid (NA), insulin, and the A1 adenosine receptor agonist SDZ WAG 994 with plasma glucose clamped at basal levels. Lowering plasma FFA with these agents all increased plasma corticosterone, but not catecholamine, within 1 h, accompanied by increases in plasma ACTH. These increases in ACTH or corticosterone were abolished when falls in plasma FFA were prevented by Intralipid during NA or insulin infusion. In addition, the NA-induced increases in plasma ACTH were completely prevented by administration of SSR149415, an arginine vasopressin receptor antagonist, demonstrating that the hypothalamus is involved in these responses. Taken together, the present data suggest that the brain may sense a fall in plasma FFA levels and activate the hypothalamic-pituitary-adrenal axis to increase plasma ACTH and corticosterone, which would help restore FFA levels. Thus, the brain may be involved in the sensing and control of circulating FFA levels.     

Abnormalities of skeletal muscle metabolism in patients with chronic heart failure: evidence that they are present at rest.

OBJECTIVE: To investigate abnormalities of skeletal muscle metabolism in patients with congestive heart failure. SETTING: A university teaching hospital. METHODS: 43 patients (22 New York Heart Association (NYHA) grade II, 21 grade III) and 10 controls were studied. A forearm model of muscle metabolism was used, with a cannula inserted retrogradely into an antecubital vein of the dominant forearm. Maximum voluntary contraction (MVC) was measured using handgrip dynamometry. Subjects performed handgrip exercise, 5 s contraction followed by 5 s rest for 5 min at 25%, 50%, and 75% of MVC or until exhaustion. Blood was taken at rest and 0 and 2 min after exercise for measurement of lactate and ammonia. After 30 min the procedure was repeated with fixed workloads of 7 kg, 14 kg, and 21 kg. RESULTS: MVC (kg, mean (SEM)) was lower in patients than in controls (control 42.45 (2.3); NYHA II 34.13 (1.3), P = 0.003; NYHA III 33.13 (1.94), P = 0.008). Resting lactate (mmol/l) was higher in patients than controls (control 0.65 (0.06); NYHA II 0.84 (0.08), P = 0.13; NYHA III 1.18 (0.1), P = 0.002). Resting ammonia (mumol/l) was higher in NYHA III (65.7 (6.0)) than in NYHA II (48.0 (3.7), P = 0.016); no difference was found between controls (48.0 (7.1)) and patients. The overall lactate and ammonia response to exercise was greater in NYHA III than in NYHA II and controls (P < 0.05). At volitional exhaustion, peak lactate (mmol/l: NYHA III 3.31 (0.26); NYHA II 2.56 (0.16); controls 2.71 (0.22); P = 0.022 NYHA III v NYHA II) and ammonia (mumol/l: NYHA III) 126.4 (8.97); NYHA II 92.9 (7.23); controls 109 (16.3); P = 0.006 NYHA III v NYHA II) were higher in severe congestive heart failure. CONCLUSIONS: Skeletal muscle metabolism is abnormal at rest in congestive heart failure. During exercise, the degree of metabolic abnormality is related to the symptomatic status of the patient.     

Further evidence against dopamine deficiency as the cause of inappropriate gonadotropin secretion in patients with polycystic ovary syndrome

Previous studies suggested that a relative dopamine (DA) deficiency may explain the altered LH secretory dynamics that occur in patients with polycystic ovary syndrome (PCO). These studies included findings of decreased urinary excretion of homovanillic acid (HVA), a metabolite of DA, and increased urinary excretion of 3-methoxy-4-hydroxyphenylglycol (MHPG), the major brain metabolite of norepinephrine. To further explore the role of DA in these patients, disulfiram (250 mg) was administered daily for 2 weeks to alter the conversion of DA to norepinephrine and to increase both peripheral and central DA in patients with PCO and in normal women. LH pulse frequency and amplitude and the serum LH response to GnRH were assessed before and during disulfiram administration. A dopaminergic effect during disulfiram administration was evidenced by a decrease in serum PRL in the PCO patients and an increase in urinary HVA excretion and a decrease in the ratio of 3-methoxy-4-hydroxyphenylglycol to HVA in urine in both groups (all P less than 0.05). This increase in DA did not significantly alter the serum estrogen level, the mean serum LH level, LH pulse amplitude, or serum LH responses to GnRH in either the PCO patients or the normal women. These data suggest that increasing endogenous DA does not correct the inappropriate gonadotropin secretion characteristic of PCO and places further doubt on the importance of DA in explaining the altered LH secretory dynamics in these patients.     

Suppression of the positive feedback of estradiol benzoate on gonadotropin secretion by an inhibitory analog of luteinizing hormone-releasing hormone (LRH) in oophorectomized rhesus monkeys: evidence for a necessary synergism between LRH and estrogens

Estrogens have a biphasic effect on gonadotropin (FSH and LH) secretion in human and nonhuman primates. Recent studies suggest that estrogens induce negative as well as positive feedback on gonadotropin secretion by a direct action at the pituitary gland. To further investigate the mechanism by which estrogens modulate gonadotropin secretion, the following experiments were performed. Oophorectomized rhesus monkeys were injected with estradiol benzoate (EB) at a dose of 40 micrograms/kg sc at zero time (controls). Three groups of animals received, additionally, an inhibitory analog of LRH, [Ac-D-p-Cl-Phe1,2, D-Trp3,D-Arg6,D-Ala10]LRH (200 micrograms, sc, daily) at 1) -48 to 48 h, 2) 0-48 h, and 3) 24-48 h from EB treatment, respectively. Blood was collected at -72, -48, -24, and 0 h, every 6 h from 6-84 h, and 96 and 116 h after EB; FSH and LH levels were measured by RIA. Control animals had a 70-80% decrease in baseline FSH and LH levels during the first 24 h after EB injection, followed by a sharp rise to 60-80% above basal levels 42-48 h post-EB. In contrast, animals that received the inhibitory analog of LRH in different regimens had more pronounced and prolonged negative feedback of EB on gonadotropin concentrations. The positive feedback release of FSH and LH was significantly diminished or delayed compared to control values. The results of this study indicate that this inhibitory analog of LRH potentiates the negative feedback and suppresses the positive feedback of EB on gonadotropins, suggesting that estrogens induce the secretion of FSH and LH through a mechanism that involves the action of LRH at the gonadotroph.     

Inhibition of P450 aromatase enhances gonadotropin secretion in early and midpubertal boys: evidence for a pituitary site of action of endogenous E

In early pubertal boys, E concentrations are very low. We studied the role and site of action of endogenous E in the regulation of gonadotropin secretion in early and midpubertal boys by inhibiting the action of E with a potent and specific P450 aromatase inhibitor, letrozole. A total of 35 boys who were referred to us because of suspicion of delayed puberty were included in the study. The boys were in either early or midpuberty, and they composed 3 groups: 10 boys did not receive any treatment, 12 boys received T alone, and 13 boys received T and letrozole. In the untreated group during the 5-month follow-up, no changes were observed in 17beta-E2, T, basal gonadotropin, or inhibin B concentrations or in the GnRH-induced gonadotropin responses. In the T-treated group during the 5-month treatment, the T concentration increased by 55% (P < 0.05), and the 17beta-E2 concentration increased by 130% (P < 0.02). Concurrently, basal gonadotropin concentrations were suppressed, but the GnRH-induced gonadotropin responses and the inhibin B concentration remained unchanged. In the T- plus letrozole-treated group during the 5-month treatment, an increase in T concentration of 606% was observed (P < 0.001), but the 17beta-E2 concentration remained unchanged. The changes in the 17beta-E2 concentration within 5 months in the untreated and the T- plus letrozole-treated groups were different (P < 0.02), indicating significant inhibition of endogenous E synthesis during letrozole treatment. During the T plus letrozole treatment, basal gonadotropin concentration, the GnRH-induced LH response, and inhibin B concentration increased, and the GnRH-induced FSH response did not change significantly. Serum nocturnal gonadotropin pulses were determined in 5 boys treated with T and in 5 boys treated with T plus letrozole. In the T- plus letrozole-treated group, the nocturnal LH pulse amplitude increased, and the LH pulse frequency and interpulse interval remained unchanged. In conclusion, in early and midpubertal boys, suppression of the action of E by the P450 aromatase inhibitor increased LH concentration, LH pulse amplitude, and the GnRH-induced LH response, which indicates that in boys during early and midpuberty, endogenous E regulates LH secretion at the site of the pituitary.     

Isolation of a T-cell clone that reacts with both antigen and anti-idiotype: evidence for anti-idiotype as internal image for antigen at the T-cell level.

T-cell lines were derived from ferredoxin nonresponder B10.D2 mice that share an idiotype expressed by a monoclonal antibody (Fd-B2) with specificity for one of the two major antigenic determinants (the C determinant) of the antigen. The T-cell line and T-cell clones derived from it release interleukin 2 not only in the presence of anti-Fd-B2 idiotype antibody but in the presence of ferredoxin. The line was shown to be major histocompatibility complex-restricted in that it would respond to the anti-idiotype and antigen only in the context of presentation by cells of the H-2d haplotype. This observation also establishes that the nonresponder status of H-2d animals cannot be attributed to a lesion at the level of antigen presentation. Analysis of the fine specificity of one idiotypic clone showed that it responded only to the anti-idiotype or products of the antigen containing the C determinant, since enzymatically degraded peptides devoid of this determinant did not stimulate these cells. Furthermore, it was found that presentation of both the antigen and the anti-idiotype to the specific clone could be blocked by the Fd-B2 monoclonal antibody.     

Identification of the second gonadotropin-releasing hormone in chicken hypothalamus: evidence that gonadotropin secretion is probably controlled by two distinct gonadotropin-releasing hormones in avian species.

A new peptide having gonadotropin-releasing activity distinct from the known luteinizing hormone-releasing hormones ( LHRHs ) has been identified in a chicken hypothalamic extract. The existence of [Gln8]LHRH in avian hypothalamus has been reported previously. The new molecular species of gonadotropin-releasing activity, named chicken gonadotropin-releasing hormone II (chicken GnRH-II), has been isolated recently in a yield of 7 micrograms, starting from 10,000 chicken hypothalami. Structural analyses have been performed on the peptide fragments derived from chymotryptic and thermolytic digests of chicken GnRH-II by amino acid analyses and terminal analyses. The full sequence of chicken GnRH-II has been determined to be: pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. A synthetic decapeptide with the above sequence was verified to be chromatographically identical to natural chicken GnRH-II. For further structural confirmation, chymotryptic and thermolytic peptides from synthetic and natural chicken GnRH-II also were identified. Thus, the structure of chicken GnRH-II has been definitely established. The gonadotropin-releasing potency of chicken GnRH-II was about 32% of that of mammalian LHRH and 8 times more potent than chicken LHRH, as estimated from the bioassay with rat anterior pituitary cells. Our results indicate that gonadotropin secretion is probably controlled by two distinct GnRHs , at least in avian species.