Variable transcriptional activity and ligand binding of mutant beta 1 3,5,3'-triiodothyronine receptors from four families with generalized resistance to thyroid hormone.

Mutations in the gene encoding the human beta 1 T3 receptor (hTR beta 1) have been associated with generalized resistance to thyroid hormone (GRTH). We measured the T3-binding affinity and transcriptional regulatory capacity of the mutant hTR beta 1 from four unrelated kindreds with GRTH. These mutations are contained in different functional regions of the ligand-binding domain. The T3 affinity of the mutant receptors correlated well with the degree of impairment of their trans-activating function in a transient cotransfection system in HeLa cells; two mutant receptors with undetectable ligand affinity showed no transcriptional activity, whereas the two other mutants characterized by a 2- and 5-fold reduction in T3 affinity required 5- and 15-fold higher T3 concentrations for half-maximal activity in the cotransfection assay, respectively. All of the mutant hTR beta 1s were able to inhibit the function of transfected normal hTR beta 1 and endogenous retinoic acid receptor in activating a palindromic positive T3 response element (TRE). In the partially functional mutants this dominant negative effect could be completely reversed by increased T3 concentrations. The dominant negative potency did not depend on the type of TRE used; mutant hTR beta 1s were able to inhibit normal receptor function to the same degree on a dimer-permissive palindromic TRE as on a nondimer-permissive inverted repeat of two identical half-sites separated by five spacer bases. However, the dominant negative potency was dependent on the absolute amount of receptor expression vector transfected. The expression of normal and mutant hTR beta 1 was assessed by immunocytochemistry. The hTR beta 1 protein levels in HeLa cells paralleled the amount of transfected expression vector. Moreover, all the mutant receptors were properly expressed in the nuclei of the transfected cells. These data suggest that different mutations in the ligand-binding domain of the human hTR beta 1 result in a variable degree of functional impairment, which may partially explain the phenotypic differences between kindreds with GRTH. Our findings suggest that competition for binding to the TRE and possibly the binding of limiting accessory factors may be more important in mediating the dominant negative effect than the formation of normal/mutant T3 receptor dimers.

A homozygous deletion in the c-erbA beta thyroid hormone receptor gene in a patient with generalized thyroid hormone resistance: isolation and characterization of the mutant receptor.

Different point mutations have been identified in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene that are associated with variant phenotypes of generalized thyroid hormone resistance (GTHR). In most cases of GTHR, heterozygotes are affected; a single mutant allele results in the inhibition of the function of normal thyroid hormone receptors. We report here a novel genetic abnormality, a 3-basepair (bp) deletion in the T3-binding domain of the beta-receptor in a kindred, S, with GTHR. One patient, S1, was the product of a consanguineous union of two heterozygotes and was homozygous for this defect. Heterozygotes from kindred S harbored a CAC deletion at nucleotides 1295-1297, which resulted in the deduced loss of amino acid residue threonine at codon 332, and they displayed elevated free T4 levels and inappropriately normal TSH levels characteristic of other kindreds with GTHR. However, patient S1, who had two mutant alleles, had markedly elevated TSH and free T4 levels and displayed profound abnormalities in brain development and linear growth. A fibroblast c-erbA beta cDNA extending from codon 175 to stop codon 457 was cloned from patient S1, sequenced, and used to create a full-length mutant cDNA. The kindred S mutant receptor was synthesized in vitro and did not bind T3. This mutant receptor did bind with similar avidity as the wild-type human beta-receptor to thyroid hormone response elements of the human TSH beta (-12 to 43 bp) and rat GH (-188 to -160 bp) genes. Kindred S showed the effect in man of heterozygous and homozygous expression of a dominant negative form of c-erbA beta.

Sex differences in growth hormone (GH) secretion by rats administered GH-releasing hexapeptide.

The purpose of this study was to compare GH secretion after the administration of GH-releasing hexapeptide (GHRP-6) in conscious male and female rats. Plasma GH was significantly elevated in female rats (six of six) compared to male rats (three of six) 15 min after administration of a single sc injection of GHRP-6 (0.5 mg/kg). In male rats, GHRP-6 administration was associated with suppression of episodic GH secretion and desensitization to a second injection administered 6 h later, whereas in female rats, GH secretion occurred after both GHRP-6 injections. After 14 consecutive days of administering GHRP-6 twice per day, mean plasma GH concentrations in males decreased from 110 +/- 91 to 2.8 +/- 0.6 ng/ml (P less than 0.05) and in females increased from 170 +/- 53 ng/ml to 361 +/- 81 ng/ml (P less than 0.05). Desensitization to GHRP-6 in conscious male rats was not observed in pentobarbital-anesthetized male rats, suggesting that GHRP-6 administration enhanced somatostatin release in the conscious state. After 14 consecutive days of GHRP-6 administration, the mean pituitary GH concentration in female rats was significantly lower than that in male rats (5.1 +/- 0.2 vs. 12.9 +/- 1.2 micrograms/mg, respectively). Lower pituitary GH concentrations in females correlated with higher GH secretion after GHRP-6 administration. Desensitization to GHRP-6 in male rats is attributed to neurohumoral factors producing their unusual pattern of episodic GH secretion, and the response is probably not typical of other species.

Role of selected endogenous peptides in growth hormone-releasing hexapeptide activity: analysis of growth hormone-releasing hormone, thyroid hormone-releasing hormone, and gonadotropin-releasing hormone.

The purpose of this study was to evaluate the contribution of endogenous GH-releasing hormone (GHRH) to exogenous GH-releasing hexapeptide (GHRP-6) activity, and to determine whether TRH or GnRH are endogenous analogs of GHRP-6. The activity of GHRP-6, a synthetic GH secretagogue, was significantly attenuated in rats administered GHRH antiserum or alpha-methyl-rho-tyrosine to reduce endogenous GHRH concentrations, and also in rats administered 5-50 micrograms/kg of [N-Ac-Tyr1,D-Arg2]-GRF 1-29 amide to block pituitary GHRH receptors. However, GHRP-6 activity was potentiated in rats administered 150 micrograms/kg [N-Ac-Tyr1,D-Arg2]-GRF 1-29 amide, presumably due to partial agonist activity of the GHRH receptor antagonist at the higher dose. These data show that endogenous GHRH contributes to full expression of exogenous GHRP-6 activity in vivo. Like TRH, a subthreshold dose of GHRP-6 was significantly more effective in hypothyroid rats than in euthyroid rats. However, suprathreshold doses of GHRP-6 were less effective in hypothyroid rats. Unlike TRH, GHRP-6 had no effect on GH and prolactin release from GH3 cells, and TRH and GnRH were poor competitors for 3H-GHRP-6 binding sites on pituitary membranes. A GnRH receptor antagonist did not block GHRP-6 activity in vivo, and GnRH administered alone or in combination with GHRP-6, did not stimulate GH release. The results of this study suggest that synergy between GHRH and GHRP-6 seen in pharmacological studies is physiologically relevant, and that TRH and GnRH are not endogenous analogs of GHRP-6.

Effects of coadministered growth hormone (GH)-releasing hormone and GH-releasing hexapeptide on maladaptive aspects of obesity in Zucker rats.

The purpose of this study was to determine the effect of chronic pharmacological stimulation of the pituitary gland on GH hyposecretion and other maladaptive aspects of obesity. Obese Zucker rats were coadministered GH-releasing hormone (GHRH; 3 micrograms/kg) and GH-releasing hexapeptide (GHRP-6; 300 micrograms/kg), a potent combination of synergistic GH secretagogues, once daily for 60 consecutive days. Although pituitary weights and GH concentrations were higher in obese rats administered the peptides than in obese rats administered saline, stimulated GH secretion was lower in obese rats than in lean rats. However, compared to those in lean rats, plasma insulin-like growth factor-I and insulin concentrations were higher in the obese rats regardless of treatment. The GH secretagogues did not alter food intake or body weight gain in sexually mature obese rats, whereas body weight gain was significantly increased when they were administered to prepubertal obese rats. Although glucose tolerance was impaired in both groups of obese rats, it improved in obese rats administered GHRH and GHRP-6 compared to that in obese rats administered saline. On the other hand, plasma cholesterol concentrations were elevated in obese rats administered the GH secretagogues but not saline. In conclusion, the results of this study suggest that hyposensitivity to GHRH and GHRP-6 in obese Zucker rats results from high concentrations of plasma insulin-like growth factor-I that negatively feedback on stimulated GH secretion. Nonetheless, daily episodes of endogenous GH secretion resulting from chronic coadministration of GH secretagogues significantly influenced the pituitary gland as well as lipid and carbohydrate metabolism.

Sertoli cell maturation is impaired by neonatal passive immunization with antiserum to luteinizing hormone-releasing hormone.

Male rats treated with a single injection of antiserum to LHRH (LHRH-AS) at 5 days of age have small testes as adults. In the present investigation, the serial maturation of the hypothalamic-pituitary-gonadal axis was studied in young male rats passively immunized with LHRH-AS. Testicular and epididymal weights, serum androgen and gonadotropin levels, testicular receptors for human CG (hCG), and androgen binding protein (ABP) concentrations in serum, testis, and epididymis were compared in developing animals treated with a single ip injection of LHRH-AS or normal rabbit serum. Rats treated with LHRH-AS had lower serum concentrations of ABP at all ages; the highest levels were on days 22-24, which were several days later than controls. Testicular weight was about 60% that of the control at all ages from 10-90 days. A reduction in epididymal weight to 80% that of the control was seen only in adults at days 60 and 90. Testicular ABP content increased steadily with age, but its concentration peaked at day 17 for controls and day 22 for LHRH-AS treated animals. Both testicular and epididymal ABP content were commensurate with testicular weight in controls and treated rats through day 45. Similarly, hCG-receptor content and concentration increased steadily with age, but differences between control and treated groups paralleled testicular weight. These results suggest an effect of LHRH blockade at a critical period which impairs early testicular growth and causes a permanent reduction in growth. Sertoli cell function and hCG-receptor appearance are impaired in proportion to this reduction.

Blunted prolactin response to thyrotropin-releasing hormone stimulation in cystinotic children receiving cysteamine.

We investigated whether long term cysteamine therapy in cystinotic children altered their basal and stimulated serum PRL levels. Five subjects who had normal plasma PRL responses to TRH stimulation before cysteamine treatment each had a lower basal PRL level and a blunted PRL response during long term (19-59 months) cysteamine therapy. A blunted PRL response was not found in 12 cystinotic subjects of the same age and stage of disease who had not received cysteamine. The effect on PRL release was not found in 2 subjects who received short term (1-2 weeks) treatment with full-dose cysteamine (50 mg/kg . day). The TSH response to TRH stimulation was not blunted during long term cysteamine therapy. These findings suggest that cysteamine alters PRL secretion in humans.

Pituitary resistance to thyroid hormone in cystinosis.

Eleven children with nephropathic cystinosis without clinical features of hyperthyroidism or hypothyroidism had elevated serum levels of immunoreactive TSH. The mean TSH level (+/- SE) was 37.4 +/- 12.3 microM/ml. Serial determinations of thyroid function during 1 yr were: mean (+/- SE) serum T4, 10.8 +/- 0.7 microgram/dl; free T4, 2.1 +/- 0.2 ng/dl; and T3, 239 +/- 6 ng/dl. After 500 microgram TRH iv, the peak TSH level exceeded 100 microU/ml in 6 of 10 patients, whereas T3 responses were variable. Studies of parameters influenced by thyroid hormone, including red cell sodium content, serum cholesterol, and 24-h urinary hydroxylysine excretion, were consistent with euthyroidism. On the other hand, the mean pulse wave arrival time was significantly reduced, consistent with hyperthyroidism. Three control patients, 2 with Lowe's syndrome and 1 with benign cystinosis, had normal thyroid studies. Eight of the patients were given either exogenous L-T4 or T3 in doses which were increased at weekly intervals. The serum TSH concentrations were suppressed to normal only after elevation of serum levels of thyroid hormones and with high exogenous thyroid replacement doses. The data suggest abnormal pituitary resistance to feedback by thyroid hormone in patients with cystinosis. We believe this to be the first description of the association of a heritable metabolic disease with such pituitary resistance.

Preservation of dopaminergic and alpha-adrenergic function in children with growth hormone neurosecretory dysfunction.

The integrity of dopaminergic and alpha-adrenergic neurotransmitter regulation of GH secretion was examined in children with decreased GH secretion. Children with GH neurosecretory dysfunction (GHND; n = 16) those with classical GH deficiency (n = 9), and short but otherwise normal children (n = 12) underwent 24 h GH studies (blood sampling every 20 min for 24 h) and provocative tests using arginine, insulin hypoglycemia, L-dopa (dopaminergic) and clonidine (alpha-adrenergic), and GH-releasing hormone (GHRH). GHND was defined as children with height in the first percentile or below, growth velocity of 4 cm/yr or less, low plasma somatomedin-C for age, delayed skeletal age by 2 or more yr, peak serum GH responses to any one (or more) provocative test of 10 ng/ml or more, and mean 24-h GH concentration below 3 ng/ml. GHND and GH-deficient children had reduced endogenous GH secretion, expressed as mean serum 24-h GH concentration [1.6 +/- 0.1 (+/- SEM) and 2.1 +/- 0.1 vs. 6.1 +/- 0.5 ng/ml (GH-deficient and GHND vs. normal, respectively); P less than 0.01]. the mean peak serum GH levels after arginine [8.2 +/- 2.0 vs. 20.8 +/- 6.6 ng/ml (GHND vs. normal); P less than 0.05] and insulin [9.3 +/- 1.0 vs. 16.2 +/- 1.7 ng/ml (GHND vs. normal); P less than 0.01) were lower in GHND children. The mean peak responses after L-dopa [13.4 +/- 3.4 vs. 14.6 +/- 4.7 ng/ml (GHND vs. normal); P = NS] and clonidine [19.0 +/- 2.2 vs. 23.3 +/- 3.8 ng/ml (GHND vs. normal); P = NS] were preserved in GHND children. In GH-deficient children, mean peak serum GH concentrations after all four provocative tests were low (arginine, 2.7 +/- 0.8; insulin, 2.6 +/- 0.8; L-dopa, 3.0 +/- 0.9; clonidine, 3.4 +/- 1.0 ng/ml; all P less than 0.01 vs. normal). The mean peak serum GH concentration after GHRH was blunted in GH-deficient children (9.1 +/- 1.7 ng/ml) compared to those in GHND (32.9 +/- 8.5 ng/ml) and normal (43.2 +/- 6.4 ng/ml) children (P less than 0.01). The area under the GH curve after GHRH stimulation was greater for normal than GHND children (P less than 0.05). These data demonstrate preservation of dopaminergic and alpha-adrenergic neurotransmitter pathways in GHND children. They further suggest a defect in the release of pituitary GH secondary to an abnormality in alternative neurotransmitter pathways resulting in decreased GHRH and/or increased somatostatin secretion.

Growth hormone (GH) provocative testing frequently does not reflect endogenous GH secretion.

GH secretion was studied in 73 children with classical GH deficiency or GH neurosecretory dysfunction (GHND), intrinsic short stature, or normal stature. The GH-deficient group was defined by a peak GH secretory response below 10 ng/ml to all provocative tests (arginine, L-dopa, insulin hypoglycemia, and clonidine). GHND was defined by a mean serum 24-h GH concentration below 3 ng/ml, with a normal response (greater than or equal to 10 ng/ml) to provocative testing. Twenty-one GH-deficient children, 21 children with GHND, and 18 short control children underwent provocative GH testing and a 24-h study with GH sampling every 20 min. A group of 13 normal stature control children also underwent 24-h GH sampling. The mean stimulated peak serum GH level [4.7 +/- 0.6 (+/- SEM) ng/ml] in the GH-deficient group was significantly below that in the GHND (19.5 +/- 1.7 ng/ml) and short control groups (24.0 +/- 3.5 ng/ml; P less than 0.01). The mean 24-h serum GH concentration was reduced in GH-deficient (1.5 +/- 0.2 ng/ml) and GHND (2.0 +/- 0.1 ng/ml) children compared to those in short (5.6 +/- 0.5 ng/ml) and normal stature (5.8 +/- 0.8 ng/ml) control children (P less than 0.01). Peak GH concentrations after provocative testing correlated poorly with 24-h mean concentrations in GH-deficient, GHND, and short control children (r = 0.38, 0.23, and 0.41, respectively; P = NS for all groups). Mean serum GH concentrations from blood sampling intervals of 12 h (day/night; 0800-2000/2000-0800 h, respectively) or even 6 h (day; 0900-1500 h) were statistically different in GHND or GH-deficient groups compared to those in control children; however, there was significantly more overlap for individual children using the 6- and 12-h daytime intervals than for the 24-h data. Plasma somatomedin-C/insulin-like growth factor I correlated with mean 24-h GH concentration endogenous secretion (r = 0.7; P less than 0.001). These data suggest that provocative GH testing frequently does not correlate with endogenous GH secretion.

Hypothalamo-hypophyseal thyroid and gonadal function before and after erythropoietin therapy in dialysis patients.

We examined the effects of administration of two hypothalamic neurohormones, TRH and GnRH, for 3 days in five anemic male dialysis patients and five age-matched normal male volunteers. Patients on chronic hemodialysis have abnormal hypothalamo-hypophyseal thyroid and gonadal functions, including blunted TSH response to TRH, hyperprolactinemia, elevated basal levels of LH with exaggerated response to GnRH, and depressed FSH secretory response to GnRH. After correction of anemia with exogenous erythropoietin, these dialysis patients were given a single injection of the same hypothalamic hormones. The repeat studies after the correction of anemia showed normalization of 1) the TSH response to TRH, 2) basal GH and PRL levels, and 3) the FSH response to GnRH. Although these patients appear to have biochemical evidence of testicular failure, the gonadotropin response (FSH) to GnRH was not exaggerated. In addition, there was no increase in total T4 and free T4 after TRH administration. Although a free T3 response to TRH was present, it was remarkably blunted compared to that of controls. At the present time, it is not known whether these hormonal responses after the correction of anemia are due to better oxygenation or a trophic action of the erythropoietin.

Pooled prolactin measurements in the evaluation of short children.

PRL secretion was determined in 63 children undergoing evaluation of GH status. Children were assigned to 1 of 3 groups based on GH studies: group 1, those with abnormal GH responses to provocative testing (n = 23); group 2, children with normal GH responses to provocative testing and mean 24-h GH concentrations below 2.5 micrograms/L (n = 14); or group 3, those with normal stimulated GH secretion and mean 24-h GH concentrations of 2.5 micrograms/L or more (n = 26). Serum PRL concentrations were measured in daytime (0800-1600 h), nighttime (2200-0600 h), and 24-h pools of serum specimens obtained every 20 min over a 24-h period. Mean (+/- SD) daytime (17.5 +/- 14.3 micrograms/L) and 24-h (19.2 +/- 13.0 micrograms/L) pool PRL concentrations were significantly higher in group 1 than in group 3 (daytime, 6.7 +/- 2.3; 24 h, 10.2 +/- 2.5 micrograms/L; P less than 0.01). Mean nighttime pool PRL concentrations did not differ among groups. Mean nighttime pool PRL values were significantly higher (P less than 0.01) than daytime pool values in group 3 (nighttime pool, 13.6 +/- 3.6 micrograms/L; night to day ratio, 2.2 +/- 1.0) and group 2 (16.8 +/- 9.0 micrograms/L; night to day ratio, 2.5 +/- 1.5), but not within group 1 (21.4 +/- 13.5 micrograms/L; night to day ratio, 1.4 +/- 0.5). The mean peak and increment in PRL concentrations after an iv bolus of insulin-TSH-LHRH were not different among groups. The mean decrement in serum PRL level after L-dopa ingestion was greater in group 1 than in group 3 (P less than 0.05). Two children in group 2 and 10 in group 1 had significantly elevated daytime pool PRL concentrations (greater than 11.3 micrograms/L; 2 SD above the mean value for group 3). Two additional children in group 2 and 2 in group 1 had elevated 24-h (greater than 15.2 micrograms/L) pool PRL concentrations. One child in group 2 and 3 in group 1 had low 24-h PRL concentrations (less than 5.2 micrograms/L; less than 2 SD below the mean of group 3). Fourteen of 20 children with elevated daytime and/or 24-h pool PRL levels or low 24-h pool PRL values had structural or radiation-associated insults to the hypothalamic-pituitary axis evident in the history or with brain-imaging techniques; 1 had microphallus with panhypopituitarism and 5 children had no structural abnormalities.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of cranial radiation on hypothalamic-adenohypophyseal function: abnormal growth hormone secretory dynamics.

The function of the hypothalamic-adenohypophyseal unit was tested in 2 groups of rhesus monkeys before and at periodic intervals after the administration of 2400 and 4000 rads cranial radiation. This therapy was given in 10 fractions over a 2-week period. Plasma TSH, basally and after TRH administration, and LH and FSH, before and after gonadotropin-releasing hormone stimulation, were normal up to 1 yr after radiation. Plasma GH at the basal state and after arginine and L-dopa stimulation was also normal. An insulin tolerance test, however, demonstrated a blunted GH response at a dose (0.1 U/kg) that caused brisk stimulation of GH secretion in normal control monkeys. A larger dose of insulin (0.2 U/kg) resulted in ample secretion of GH in these animals, suggesting decreased hypothalamic sensitivity to insulin in treated animals. The measurement of GH every 20 min for 24 h in animals treated with 4000 rads showed a dramatically altered secretory pattern of GH 1 yr after radiation. GH secretory spikes were markedly decreased in both frequency and amplitude, suggesting a reduction in the normal daily production of GH.

The effects of corticotropin and growth hormone releasing hormones on their respective secretory axes in chronic hemodialysis patients before and after correction of anemia with recombinant human erythropoietin.

Endocrine abnormalities in chronic hemodialysis patients are in part corrected by control of anemia with recombinant human erythropoietin (rHu-EPO). We further examined the role of rHu-EPO in select hormonal abnormalities thought to be anemia related as well as the GH-insulin-like growth factor 1 (GH-IGF-1) axis that is abnormal in hemodialysis patients. We studied responses to the administration of two hypothalamic hormones, GHRH and ovine corticotropin-releasing hormone (CRH), in five anemic male patients on chronic hemodialysis before and after correction of the anemia with rHu-EPO. For comparison, five age-matched normal male volunteers were tested once. Anemic patients on chronic hemodialysis had high basal GH concentrations, an exaggerated GH response to exogenous GHRH, increased levels of IGF-1, and elevated levels of IGF-1 binding protein-3 in comparison to controls. ACTH response to CRH was comparable in dialysis patients and normal controls, but the cortisol response to endogenous ACTH release was prolonged. The cortisol binding globulin was similar to the controls. After correction of anemia, the basal elevation of GH was no longer present, but the exaggerated response of GH to exogenous GHRH persisted. IGF-1 and IGF-1 binding protein-3 levels remained elevated. The ACTH response to CRH, which was normal before correction of the anemia, became exaggerated in terms of elevated levels. Nevertheless, the prolonged cortisol response persisted. It appears that correction of the anemia in hemodialysis patients with rHu-EPO can partly correct perturbations in the GH secretory axis but may lead to new abnormalities in the CRH-ACTH axis.

Competitive polymerase chain reaction quantitation of c-erbA beta 1, c-erbA alpha 1, and c-erbA alpha 2 messenger ribonucleic acid levels in normal, heterozygous, and homozygous fibroblasts of kindred S with thyroid hormone resistance.

Mutations in the T3-binding domain of the thyroid hormone receptor gene c-erbA beta result in dominant negative proteins and thyroid hormone resistance syndromes. Variable clinical manifestations of resistance to thyroid hormones have been reported, including short stature and neuropsychological abnormalities. The molecular bases for heterogeneity of phenotype among and within kindreds have not been fully elucidated. Recent investigations have considered differential expression of mutant and wild-type beta 1-receptor alleles and the regulation thereof as a mechanism to explain differential sensitivity to thyroid hormones. We used reverse transcription-competitive polymerase chain reaction (PCR) to measure c-erbA beta 1, c-erbA alpha 1, and c-erbA alpha 2 mRNAs in skin fibroblasts cultured from normal subjects, heterozygotes, and a severely affected homozygous mutant of kindred S. The homozygous mutant of kindred S had severe growth and mental retardation. After reverse transcription with primers specific for each of the c-erbA mRNAs, first strand cDNAs were amplified by PCR using subtype-specific amplimers. Primer design allowed simultaneous detection of wild-type and mutant messages in heterozygous fibroblasts and showed an approximately 1:1 ratio of these mRNAs in three patients. Inclusion of competitive standard cDNAs of known concentration in the PCR reactions allowed quantitation of the absolute levels of the beta 1-, alpha 1-, and alpha 2 mRNAs by comparison of products on ethidium bromide-stained agarose gels. These studies showed no effect of the presence of the mutant beta 1-allele, as fibroblast RNA from normal subjects, heterozygotes, and the homozygote gave values of 56-184, 2.8-12, and 23-40 attomol/5 micrograms total RNA for beta 1-, alpha 1-, and alpha 2 mRNAs, respectively. We conclude that these sensitive methods allow the detection of molecular species present at levels as low as 10 molecules/cell, and that this potent dominant negative receptor does not disrupt c-erbA expression at the level of mRNA. The neuropsychological sequelae of the kindred S mutation are not due to relative overexpression of the mutant allele.

Effect of GnRH antagonist, [Ac-delta 3Pro1, pFDPhe2, DTrp3,6] GnRH, on pulsatile gonadotrop in secretion in the castrate male primate.

Three different doses of a potent antagonist to GnRH, [Ac-delta 3Pro1, pFDPhe2, DTrp3,6], GnRH were compared in adult male monkeys to determine the acute effect on pulsatile gonadotropin secretion. In accomplishing these studies, blood was drawn at 15 min intervals over 24-30 h without anesthesia using a mobile vest and tether assembly to support an indwelling cannula. After a 3 to 6 h control period, 2.0, 0.2 or 0.02 mg GnRH antagonist/kg bw in 1 ml corn oil sc, was given to castrate adult monkeys. The highest dose decreased circulating LH (by radioimmunoassay and bioassay) and FSH whereas the intermediate dose decreased LH only. The higher dose produced both a more prolonged and greater reduction in circulating gonadotropins. These data demonstrate that this GnRH antagonist can reduce serum gonadotropins both acutely and for intervals greater than 24 h.

Male sexual development in the monkey. II. Cross-sectional analysis of pulsatile hypothalamic-pituitary secretion in castrated males.

Pulsatile secretion of serum gonadotropins was studied in 16 castrated monkeys from 4 weeks of age through adult life. Animals were castrated at various ages from birth through adult life. Although some studies of the gonadotropin-secretory patterns were longitudinal in nature, most comparisons were cross-sectional. On the basis of our observations, we have arbitrarily grouped the animals into 4 developmental ages: postnatal (less than 7 months), prepubertal or juvenile (7-27 months), pubertal (28-59 months), and adult (greater than or equal to 60 months). In carrying out these studies, blood was withdrawn at 15-min intervals over 24 h without anesthesia using a mobile vest and tether assembly to support an indwelling catheter. GnRH challenge tests were done on 1 or more occasions on all animals. Plasma samples were analyzed for concentrations of FSH and LH by established RIAs and an in vitro bioassay for LH. During the frequent sampling period (24 h for all except postnatal animals), the amplitude of gonadotropin pulses was greatest in adult animals followed by postnatal and pubertal monkeys. During pubertal development, there was a marked increase in the magnitude of gonadotropin pulses, and remarkedly, there was a substantial increase in the LH bioassay: RIA (greater than 5:1) by adult life. GnRH challenge tests of gonadotropins correlated with these observations. Time series analysis was applied to the data for objective statistical characterization of cyclic patterns. Our findings can be summarized: 1) during pubertal maturation there is a change in amplitude but not frequency of gonadotropin pulses, 2) pubertal development of the hypothalamic-pituitary axis advances in the absence of gonadal feedback, and 3) there is a significant increase in the LH bioassay: RIA during pubertal development. We conclude that the castrate monkey is a valuable adjunct to direct clinical investigations of the mechanisms controlling human sexual development.

Male sexual development in the monkey. I. Cross-sectional analysis of pulsatile hypothalamic-pituitary-testicular function.

Pulsatile secretion of serum gonadotropins and testosterone was studied in 46 monkeys of varying ages from 9 days of age through adult life. Although some of the hormonal analysis was longitudinal in nature, most comparisons were cross-sectional. On the basis of pulsatile secretory patterns, hCG and GnRH stimulation, skeletal age, testicular volume, and histology, we have arbitrarily defined four developmental age groups: postnatal (less than 7 months), prepubertal or juvenile (7-27 months), pubertal (28-59 months), and adult (greater than or equal to 60 months). In accomplishing the pulsatile studies, blood was withdrawn at 15-min intervals over 24 h without anesthesia using a mobile vest and tether assembly to support an indwelling cannula. GnRH and hCG challenge tests were done on one or more occasions on all animals. Plasma samples were analyzed for concentrations of FSH, LH, testosterone, dihydrotestosterone and delta 4-androstenedione by established RIAs and an in vitro bioassay for LH. During the frequent sampling period of 24-h duration for all except postnatal animals, testosterone pulses of large amplitude (up to 8-fold) occurred in postnatal, pubertal, and adult animals. Pulsatile gonadotropin secretion was seen at all ages; however, the highest pulses (up to 15-fold) occurred in prepubertal animals even though this was an infrequent occurrence. Time series analysis techniques were applied for objective statistical characterization of cyclic patterns. Basic rhythms corresponding to 50- to 90-min frequency cycles in gonadotropin secretion were identified. Substantive differences between LH concentrations by bioassay and RIA were seen infrequently. Our findings illustrate that: 1) circulating gonadotropin and testosterone pulses change in amplitude but not necessarily frequency during pubertal development, and 2) primate models are a useful paradym for the longitudinal study of human male sexual development. We conclude that where direct human investigation may be limited, much can be learned by study of these primate surrogates.

Homozygosity for a dominant negative thyroid hormone receptor gene responsible for generalized resistance to thyroid hormone.

Generalized resistance to thyroid hormones (GRTH) commonly results from mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. We have reported on a novel deletion mutation in c-erbA beta in a kindred, S, with GRTH. One patient from this kindred was the product of a consanguineous union from two affected members and was homozygous for the beta-receptor defect. This patient at 3.5 weeks of age had unprecedented elevations of TSH, free T4, and free T3 (TSH, 389 mU/L; free T4, 330.8 pmol/L; free T3, 82,719 fmol/L). He displayed a complex mixture of tissue-specific hyperthyroidism and hypothyroidism. He had delayed growth (height age, 1 3/12 yr at chronological age 2 9/12 yr) and skeletal maturation (bone age, 4 months), and developmental delay (developmental age, 8 months), but he was quite tachycardic. The homozygous patient of kindred S is markedly different from a recently reported patient with no c-erbA beta-receptor. This difference indicates that a dominant negative form of c-erbA beta in man can inhibit at least some thyroid hormone action mediated by the c-erbA alpha-receptors.

Effects of stimulated growth hormone secretion on age-related changes in plasma cholesterol and hepatic low density lipoprotein messenger RNA concentrations.

Growth hormone (GH) secretion declines during aging. Since GH alters plasma cholesterol (PC) concentrations, it was of interest to determine how GH secretagogues affect age-related hypercholesterolemia. Fischer 344 rats (3 and 14 months old) were co-administered (s.c.) GH releasing hormone (3 micrograms/kg; GHRH) and GH releasing hexapeptide (100 micrograms/kg; GHRP) for 120 consecutive days. Aging was associated with a progressive increase in PC, which was reduced in rats administered GHRH and GHRP compared to those administered vehicle, i.e. changes in PC during the study were 26.5 +/- 1.2 mg/dl vs. 40.1 +/- 0.9 mg/dl (P < 0.05) in the younger rats and 17.6 +/- 2.3 mg/dl vs. 31.6 +/- 5.3 mg/dl (P < 0.05) in the older rats, respectively. The lower concentrations of PC in GH secretagogue-treated older rats were associated with higher mean concentrations of hepatic LDL receptor mRNA (1.27 +/- 0.4 vs. 0.4 +/- 0.1; P < 0.05) but not cholesterol 7-alpha hydroxylase mRNA. Although GH secretagogue treatment was also associated with lower plasma cholesterol in the younger rats, it was not accompanied by quantitative changes in mean group concentrations of hepatic LDL receptor mRNA. Instead, daily administration of GHRH and GHRP in the younger rats correlated with a significant reciprocal relationship (P < 0.05) between PC and hepatic LDL receptor mRNA for individual group members. The results of this study suggest that reduced GH secretion during aging contributes, at least in part, to a progressive increase in plasma cholesterol that can be partially prevented with GH secretagogues. Furthermore, the effects on PC may result from GH-mediated, qualitative and quantitative changes in hepatic LDL receptor mRNA that increase receptor-mediated cholesterol clearance.