Long-term results of long-acting luteinizing-hormone-releasing hormone agonist in central precocious puberty.

Thirty children with precocious puberty (24 girls aged 6.5 +/- 2.3 years and 6 boys aged 7 +/- 2.9 years) were treated over 5 years with Decapeptyl. In girls, the menses disappeared, breast enlargement regressed, and uterus and ovary sizes returned to prepubertal values. In boys, a significant decrease of testicular size was observed. Plasma levels of estradiol and testosterone, and basal and post-luteinizing hormone (LH)-releasing hormone (LHRH) LH and follicle-stimulating hormone (FSH) remained in the prepubertal range. Growth velocity decreased after 1 year from 9.7 +/- 3.5 to 5.5 +/- 1.3 cm/year, while the height age/bone age ratio was normalized in both sexes after 3 years. In 15 girls, Decapeptyl was interrupted after 2.3 years. During those 2.3 years, bone age increased from 11.6 +/- 0.8 to 12.5 +/- 0.7 years with a growth velocity of 5.3 +/- 1.8 cm/year. During the year following interruption, height increased from 152.2 +/- 4.9 to 157.7 +/- 4.9 cm (growth velocity 5.5 cm/year) and bone age from 12.5 +/- 0.7 to 13.5 +/- 0.6 years. One year after treatment, plasma levels of estradiol were 106.7 +/- 84.7 pg/ml, of LH, 25.5 +/- 17.6 mIU/ml, and of FSH, 10.8 +/- 5.9 mIU/ml. Menses appeared in 13 girls. Moreover, 18 months after interruption, bone age was 13.9 +/- 0.6 years and height 159.5 +/- 5.2 cm, being significantly superior to the final height of a historical control group: 151.5 +/- 4.8 cm (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of the antiprogestin, RU 486, on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.     

Effects of season and testosterone treatment on gonadotrophin secretion and pituitary responsiveness to gonadotrophin-releasing hormone in castrated Romney and Poll Dorset rams.

In castrated rams (Romney and Poll Dorset, n = 8 for each breed), inhibition by testosterone treatment (administered via Silastic capsules) of luteinizing hormone (LH) pulse frequency, basal and mean LH concentrations, mean follicle-stimulating hormone (FSH) concentration, and the peak and total LH responses to exogenous gonadotrophin-releasing hormone (GnRH) were significantly (P less than 0.01) greater during the nonbreeding than during the breeding season. Poll Dorset rams were less sensitive to testosterone treatment than Romney rams. In rams not receiving testosterone treatment, LH pulse frequency was significantly (P less than 0.05) lower during the nonbreeding season than during the breeding season in the Romneys (15.8 +/- 0.9 versus 12.0 +/- 0.4 pulses in 8 h), but not in the Poll Dorsets (13.6 +/- 1.2 versus 12.8 +/- 0.8 pulses in 8 h). It is concluded that, in rams, season influences gonadotrophin secretion through a steroid-independent effect (directly on hypothalamic GnRH secretion) and a steroid-dependent effect (indirectly on the sensitivity of the hypothalamo-pituitary axis to the negative feedback of testosterone). The magnitude of these effects appears to be related to the seasonality of the breed.     

Advanced carcinoma of the prostate: treatment with a gonadotrophin releasing hormone agonist.

Ten patients with advanced progressive adenocarcinoma of the prostate were treated with a long acting analogue of gonadotrophin releasing hormone. Eight of these patients responded to treatment in terms of pain relief and clinical regression of tumour. Serum gonadotrophin and testosterone concentrations were significantly suppressed by the end of the second week of treatment, testosterone concentrations being comparable with those achieved by castration. The two patients who failed to respond had both relapsed previously when receiving conventional treatment, and neither showed any endocrine response to the analogue. Superagonists of gonadotrophin releasing hormone may be the treatment of choice in adenocarcinoma of the prostate, but further trials are required to establish long term safety and efficacy.     

Use of quin 2 to measure calcium concentrations in ovine anterior pituitary cells and the effects of quin 2 on secretion of growth hormone and prolactin

Intracellular free Ca2+ concentrations [Ca2+]i were measured in ovine anterior pituitary cells using the quin 2 technique. Thyrotropin-releasing hormone (TRH) increased, dopamine decreased and growth hormone-releasing hormone (GHRH) had no detectable effect on [Ca2+]i. Loading the cells with quin 2, at an intracellular concentration less than that used during calcium determination, reduced both basal growth hormone (GH) and (to a small extent) prolactin secretion. Loading cells with quin 2 also markedly reduced GHRH-stimulated GH secretion. However, TRH-stimulated prolactin secretion was 3-times basal irrespective of quin 2 loading. The results indicate that the use of quin 2 to measure [Ca2+]i in some cell types may be complicated by actions of quin 2 on cellular function.     

Guanidination of ovine luteinizing hormone and effects on activity.

The free amino groups in oLH, oLHalpha and oLHbeta were guanidinated by O-methylisourea. The epsilon-NH2 groups of lysine residues reacted bo substitute these positions in the sequence with the more basic homoarginine residue. The alpha-NH2 groups did not react under the conditions used. Guanidinated oLH or the products of guanidinated oLHalpha + native oLHbeta or guanidinated oLHalpha + guanidinated oLHbeta were inactive in two bioassay systems. Native oLHalpha + guanidinated oLHbeta, however, showed potencies of 39% to 55% of that observed with the native subunit recombinant or native oLH. Possible structural implications for hormone-receptor site interactions are discussed.     

Testosterone-dependent effects of galanin on pituitary luteinizing hormone secretion in male rats

Galanin is a 29-amino-acid peptide that colocalizes with GnRH in hypothalamic neurons. High concentrations of galanin are present in portal vessel blood of both male and female rats, and galanin receptors are present on gonadotropes in both sexes. Results from studies of female rats indicate that galanin acts at the level of the pituitary to directly stimulate LH secretion and also to enhance GnRH-stimulated LH secretion. The effects of galanin on pituitary LH secretion in male rats are relatively uncharacterized; thus, the present in vivo study was conducted 1). to examine the ability of galanin to affect basal or GnRH-stimulated LH secretion in male rats and 2). to determine whether the effects of galanin on LH secretion in male rats are testosterone-dependent. All three doses of galanin used (1, 5, and 10 micro g/pulse) significantly enhanced GnRH-stimulated LH secretion in intact male rats. Only the highest dose of galanin directly stimulated LH secretion (without GnRH coadministration) in intact males. Galanin did not directly stimulate LH secretion or enhance GnRH-stimulated LH secretion in castrated male rats. In fact, the highest dose of galanin inhibited GnRH-stimulated LH secretion in castrated males. Upon testosterone replacement, the ability of galanin to directly stimulate LH secretion and to enhance GnRH-stimulated LH secretion was restored in castrated males. These results suggest a role for galanin in the regulation of LH release in male rats and demonstrate that testosterone upregulates the ability of the pituitary to respond to the stimulatory effects of galanin.     

Hypothalamic and pituitary effects of prostaglandins on ACTH secretion.

Various prostaglandins (PGs) were tested for their effects on ACTH secretion upon injection into the anterior pituitary, basomedial hypothalamus, basolateral hypothalamus, or a tail vein in anesthetized female rats. In some experiments, the PGs were injected in combination with a CRF preparation. The greatest effect seen was the stimulation of ACTH (and presumably CRF) secretion exerted at the basomedial hypothalamus. At the anterior pituitary, the PGs alone were without effect, but they did decrease the magnitude of the response to subsequent CRF.     

Effects of growth hormone-releasing factor, somatostatin and dopamine on growth hormone and prolactin secretion from cultured ovine pituitary cells

A synthetic form of human pancreatic growth hormone releasing factor (GRF-44-NH2) was shown to be a potent stimulator of growth hormone (GH) secretion and cellular cyclic AMP levels in cultured sheep pituitary cells. A small dose-dependent stimulation of prolactin secretion was also observed. Somatostatin (0.5 microM) completely blocked the maximal GRF (1 nM)-stimulated secretion without a significant effect on cyclic AMP levels. Dopamine (0.1 microM) inhibited the GRF-elevated GH secretion by 50% and lowered cyclic AMP levels by 30%. Dopamine (0.1 microM) inhibition of basal prolactin secretion was not affected by GRF (1 nM). The data support the hypothesis that cyclic AMP is involved in the action of GRF but suggest that somatostatin can inhibit GRF-induced secretion of GH independently of cyclic AMP.     

Effects of a gonadotrophin-releasing hormone antagonist on gonadotrophin secretion and gonadal development in neonatal pigs

Male (N = 8) and female (N = 8) pigs were assigned to receive saline or a potent GnRH antagonist ([Ac-D2Nal1,D4-Cl-Phe2,D-Trp3,D-Arg6, D-Ala10]- GnRH*HOAc; 1 mg/kg body weight) at 14 days of age. The GnRH antagonist caused LH to decline (P less than 0.01) from 1.7 ng/ml at 0 h to less than 0.5 ng/ml during 4-32 h in males and females. Concentrations of FSH in gilts declined slowly from 75 +/- 8 to 56 +/- 5 ng/ml (P less than 0.05) at 32 h. In males FSH was low (5.7 +/- 0.5 ng/ml) at 0 h and did not change significantly. To observe the effect of long-term treatment with GnRH antagonist, 10 male and 10 female pigs, 3 days of age, were treated with saline or 1 mg GnRH antagonist per kg body weight every 36 h for 21 days. Concentrations of LH were reduced (P less than 0.01) to 0.2-0.4 ng/ml throughout the experimental period in male and female piglets treated with GnRH antagonist. Plasma FSH increased in control females, but remained suppressed (P less than 0.001) in females treated with GnRH antagonist. Treatment with the GnRH antagonist suppressed FSH levels in males on Days 8 and 16 (P less than 0.05), but not on Day 24. Treatment of females with the GnRH antagonist did not influence (P greater than 0.10) oestradiol-17 beta concentrations. Administration of GnRH antagonist to males suppressed testosterone and oestradiol-17 beta values (P less than 0.01) and reduced testicular weight (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory effects of 1 alpha, 25-dihydroxycholecalciferol on parathyroid hormone secretion in rats

In an attempt to study the influence of vitamin D metabolites on PTH secretion, serum calcium and urinary excretion of cAMP were sequentially measured in conscious perfused rats, and the effects of a single iv injection of the metabolites on these parameters were examined. Four hours after the administration of 0.25 microgram/kg (0.6 nmol/kg, probably a physiological dose) of 1 alpha, 25-dihydroxycholecalciferol [1 alpha, 25 (OH)2D3], the urinary excretion of cAMP decreased to a level compatible with that of parathyroidectomized rats (approximately 60% of the initial value; P less than 0.05) and this level was sustained for nearly 24 h. Serum concentrations of calcium (total and ionized) did not change. In parathyroidectomized rats which were continuously infused with bovine PTH (1 U/h), the vitamin D metabolite had no significant effect on the urinary excretion of cAMP. 24 R, 25-dihydroxcholecalciferol (12.5 microgram/kg) had no significant effect either on the urinary excretion of cAMP or on serum calcium. These results suggest that in rats, a physiological dose of 1 alpha, 25(OH)2D3 inhibits PTH secretion without causing a significant rise iu serum calcium, reflecting a feed-back mechanism between active vitamin D metabolite, 1 alpha, 25(OH)2D3 and the parathyroid glands.     

Effects of pretreatment with tetradecanoyl phorbol acetate on regulation of growth hormone and prolactin secretion from ovine anterior pituitary cells

Tetradecanoyl phorbol acetate (TPA) stimulates growth hormone (GH) and prolactin secretion from ovine anterior pituitary cells. Pretreatment of the cells with TPA abolishes this effect, presumably due to down-regulation of protein kinase C. Such pretreatment did not alter effects of thyrotropin-releasing hormone or dopamine on prolactin secretion, suggesting no involvement of protein kinase C. Pretreatment with TPA attenuated actions of GH-releasing hormone on GH release (but not actions on cyclic AMP levels), possibly due to depletion of cellular stores of GH. Such pretreatment also attenuated inhibition of GH release by somatostatin, possibly due to phosphorylation of receptors or associated proteins by protein kinase C.     

Direct effects of estradiol-17 beta on the number of gonadotropin-releasing hormone receptors in the ovine pituitary

Concentrations of pituitary receptors for gonadotropin-releasing hormone (GnRH) are affected by GnRH and gonadal steroids. To test the hypothesis that estradiol-17 beta (E2) directly affects the number of GnRH receptors in the pituitary, independent of GnRH secretion, ovariectomized ewes with hypothalamic-pituitary disconnections (HPD) were given 25 micrograms (i.m.) of E2 (HPD + E2, n = 5) or oil (HPD + OIL, n = 5). Ovariectomized control ewes, with intact hypothalamic-pituitary axes (INT), also received either E2 or oil (INT + E2, n = 6; INT + OIL, n = 6). Blood samples were taken hourly for analysis of serum concentrations of luteinizing hormone (LH) from 4 h prior to until 16 h after treatment. Pituitaries were collected 16 h after treatment for analysis of GnRH receptors. Treatment with E2 increased concentrations of LH in serum beginning 12.7 +/- 0.6 h after injection in INT ewes but not in HPD ewes. Compared to INT + OIL ewes, E2 treatment increased (p less than 0.001) the number of GnRH receptors by 2.5-fold in INT ewes and by 2.0-fold in HPD ewes. These results suggest that although GnRH is necessary for secretion of gonadotropins, E2 alone can directly increase the number of GnRH receptors in the pituitary.     

Ultrastructural detection of the onset of pituitary thyrotroph sensitivity to lowered thyroid hormone concentrations in the fetal sheep

The goitrogen methylthiouracil was administered orally to pregnant ewes of known gestational ages to induce hypothyroidism in both mother and fetus. Developing pituitary thyrotrophic cells were studied using electron microscopy to detect the earliest gestational age at which morphological changes occurred in response to lowered plasma thyroid hormone concentrations. At 50 days of gestation, the pituitaries of fetuses exposed to the goitrogen were indistinguishable from untreated control glands. However, at 58 days and subsequent ages, "thyroidectomy' cells were observed in pituitaries of all hypothyroid fetuses. These findings indicate that fetal sheep pituitary thyrotrophs are sensitive to lowered thyroid hormone concentrations by 58 days of gestation, suggesting that thyroid-thyrotroph interaction exists at this early stage of development.     

Purification and characterization of high-molecular-weight forms of adrenocorticotropic hormone of ovine pituitary glands.

A highly purified preparation of high-molecular-weight adrenocorticotropic hormone (ACTH) was prepared from ovine pituitary glands by dilute acetic acid extraction, oxycellulose fractionation. Sephadex gel filtration, and affinity chromatography on immobilized alphap(1-39)ACTH antibodies. Two ACTH peptides of molecular weights of 24 000 and 34 000 were detected by sodium dodecyl sulfate-acrylamide gel electrophoresis in this preparation. It appeared that the immobilized antibodies adsorbed two forms equally well and could not distinguish between them under the conditions used. These two ACTH peptides were found to be present in crude extracts of ovine pituitary glands, indicating that they were not artifacts produced by the purification procedure. The high-molecular-weight forms of ACTH were found to be susceptible to degradation by tissue enzymes. They could be easily destroyed during the extraction, if precautions were not taken. Moreover, they were poorly adsorbed by oxycellulose which had been used for the adsorption of ACTH activity from crude preparations by most investigators. These properties probably accounted for the fact that high-molecular-weight forms of ACTH remained undetected until very recently.     

Studies on pituitary follitropin. II. Isolation and characterization of the subunits of the ovine hormone.

The α and β subunits of highly potent ovine follitropin have been isolated by dissociation in 8 m urea, pH 7.5, and chromatography on DEAE-Sephadex A25. The isolated subunits display microheterogeneity on polyacrylamide gel electrophoresis and have very low activity in follitropin-specific radioreceptor and radioimmunoassays. The tryptophan fluorescence spectra of native follitropin and the isolated β subunit are different. The recombinant of follitropin α + β subunit had the same activity as the native hormone in the radioimmunoassay, but its activity in the radioreceptor and in vivo bioassay was about 65% of the intact hormone. Substitution of the follitropin α by ovine lutropin α subunit (prepared by a method not involving urea) to form the recombinant restored full activity in all the three assays investigated. The formation of recombined hormone proceeds at a rapid rate and is almost complete by 6 h. The α and β subunits of ovine follitropin differ from each other in amino acid composition. No significant differences were apparent in their carbohydrate composition. The amino acid composition of the ovine follitropin α and lutropin α subunits are very similar. The oxidized α subunit has phenylalanine at its NH₂-terminus while aspartic acid is present at this position in the oxidized β subunit.