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Striped bass are seasonal breeding fish, spawning once a year during the spring. All 3-yr-old males are sexually mature; however, 60-64% of the fish mature earlier as 1- or 2-yr-old animals. The endocrine basis underlying early maturity in 2-yr-old males was studied at the molecular level by monitoring changes in pituitary beta FSH and beta LH mRNA levels by ribonuclease protection assay, and correlating these changes to stages of testicular development. In maturing males, the mRNA levels of beta FSH were elevated during early spermatogenesis, whereas beta LH mRNA levels peaked during spermiation. The appearance of spermatozoa in the testis was associated with a decrease in beta FSH mRNA and a rise in beta LH mRNA abundance. Immature males had lower levels of beta LH mRNA than maturing males, but there were no differences in beta FSH mRNA levels between immature and maturing males. The regulation of gonadotropin gene expression in 2-yr-old males was studied by the chronic administration of GnRH analogue (GnRHa) and testosterone (T), with or without pimozide (P) supplementation. In immature males, the combination of T and GnRHa stimulated a three- to fivefold increase in beta FSH and beta LH mRNA levels, but the same treatment had no effect on gonadotropin gene expression in maturing males. In addition, the coadministration of P to immature males suppressed the stimulatory effect of GnRHa and T on beta FSH and beta LH mRNA levels, suggesting that dopamine may have a novel role in regulating gonadotropin gene expression.
Because of some indication that FSH secretion is less dependent than LH secretion on GnRH in vivo, we performed experiments to examine the effects of a GnRH antagonist (antag) on LH and FSH secretion. We first showed that pituitary cells superfused with GnRH showed a similar pattern of suppressed secretion of both LH and FSH in response to addition of antag. In contrast, antag administration to ovariectomized rats had differing effects on LH and FSH secretion. Serum LH was suppressed in a dose-dependent fashion by 2 h (20-50% of control values). Recovery from the lower doses of antag was seen by 12 h, but the two highest doses maintained serum LH levels at 10% of control values for 72 h. In contrast, the effect on serum FSH was not manifested until 12 h. FSH was maximally decreased only to 40-60% of control values. The two highest doses maintained this effect for 72 h. These results reinforce previous suggestions that FSH secretion in vivo may occur independently of acute changes in GnRH secretion, and may have an GnRH-independent component.
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The proliferation of pituitary somatotroph cells and the synthesis and secretion of GH are under the stimulatory control of the hypothalamic peptide GHRH. GHRH is initially synthesized as pre-prohormone and then enzymatically cleaved to its mature form (44 amino acids in humans and 42 in mice). Although mutations in the GHRH receptor cause isolated GH deficiency (IGHD) both in humans and mice, mutations in the GHRH gene have never been described. To determine the consequences of generalized lack of GHRH, we have created a mouse with targeted disruption (knockout) of the GHRH gene (GHRHKO). We have substituted a portion of the gene that encodes for the initial 14 amino acids of the 1-42 GHRH with a neomycin resistance cassette. Heterozygous founder (+/-) mice were mated to obtain -/- animals. The expected Mendelian ratio was conserved (25.8% of offspring were +/+, 52.8% were +/-, and 21.4% were -/-), showing no lethality in the GHRHKO embryos. GHRHKO mice appeared normal at birth. Starting at 3 wk of age, -/- mice showed significant growth retardation. By 12 wk of age, their weight was about 60% of +/+ and +/- littermates. Growth retardation was due to IGHD, as shown by reduced pituitary GH mRNA and protein content, reduced serum IGF-I, and reduced liver IGF-I mRNA. The phenotype of the GHRHKO mice is similar to the one observed in the mouse with mutated GHRH receptor, including pituitary hypoplasia. Heterozygous mice had normal growth, although adult +/- males (but not females) had mild reduction in serum IGF-I. In conclusion, we demonstrate that ablation of the GHRH gene causes IGHD in mice. The GHRHKO mouse will be the new useful model of IGHD.
Mutations in the GnRH receptor gene (GNRHR) are a cause of idiopathic hypogonadotropic hypogonadism. We describe a normosmic female subject with congenital idiopathic hypogonadotropic hypogonadism in whom treatment with pulsatile GnRH resulted in an unusual response. The subject not only required an increased dose of pulsatile GnRH for ovarian follicular development, but LH secretion did not increase appropriately, estradiol levels remained low, and she did not ovulate spontaneously. Sequencing of the GNRHR coding sequence revealed compound heterozygous mutations leading to amino acid substitutions [N10K+Q11K] and P320L. The introduction of the P320L mutation into the GnRH receptor led to failure of detectable ligand binding and failure of stimulation of inositol phosphate production and gonadotropin subunit gene promoter activity in response to GnRH in transiently transfected cells. The [N10K+Q11K] mutation resulted in reduced binding of a GnRH agonist to 25% of the wild-type receptor. In addition, the EC(50) value for GnRH stimulation of inositol phosphate production was significantly increased, and the dose-response curves for stimulation of alpha gonadotropin subunit, LHbeta, and FSHbeta gene transcription by GnRH were similarly shifted to the right. Stimulation of FSHbeta gene transcription was more sensitive to GnRH than LHbeta for both wild-type and [N10K+Q11K] GnRH receptors, resulting in a greater loss of LHbeta stimulation than FSHbeta by the [N10K+Q11K] mutant at any given submaximal GnRH concentration. We propose that the mutations in the GnRH receptor result in a rightward shift of the dose-response curves of gonadotropin responses to pulsatile GnRH in the subject and unmask the differential sensitivities of LH and FSH to GnRH, resulting in low LH and estradiol levels despite appropriate FSH secretion and follicular growth.
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A reverse hemolytic plaque assay was used to measure the GH responses of fetal pituitary cells to GHRH, SRIH, T3 and glucocorticoids. Cells from eight human abortuses (18-22 weeks' gestation) showed accelerated plaque formation after treatment with 10(-7) mol/L GHRH-(1-44) [25.6 +/- 0.6% (+/- SE) of cells formed plaques (PFC); mean area, 14.5 +/- 2.7 X 10(4) micron2; all at 1 h], while 10(-7) mol/L SRIH-(1-28) slowed plaque formation (8.6 +/- 0.6% PFC; mean area, 4.2 +/- 0.8 X 10(4) micron2) vs. control (13.7 +/- 0.7% PFC; mean area, 5.3 +/- 0.8 X 10(4) micron2; all at 1 h). The proportion of PFC was equal in GHRH-treated and control groups by 4 h, suggesting that GHRH affects the amount of GH secreted per somatotroph rather than the number of cells that are preferentially responsive to GHRH. Qualitatively similar data were obtained using pituitary cells from four near-term rhesus fetuses. When cells were cultured in defined medium for 3 days, supplementation with T3 reduced basal GH secretion and attenuated the responses to GHRH. Culture with dexamethasone increased basal GH secretion and restored the responsiveness to GHRH. Dexamethasone also caused a shift in plaque area frequency distributions to patterns similar to those in serum-supplemented medium. We conclude that fetal somatotrophs are responsive to SRIH, GHRH, T3, and dexamethasone. Furthermore, glucocorticoids can maintain a subpopulation of fetal somatotrophs in the GHRH-responsive state.
Previous studies have shown that CRH is capable of inhibiting GH release in response to GHRH in adult subjects, and this effect appeared to be sex dependent and more pronounced in women than in men. To assess whether CRH has an inhibitory action on GH release in children also, the effects of graded doses of CRH on the GHRH-induced GH secretion were studied in three groups of prepubertal children. All subjects underwent a GHRH test (1 microgram/kg), followed, on separate occasions, by the combined administration of GHRH (1 microgram/kg) and CRH (1 microgram/kg, group A, n = 6; 1.5 microgram/kg, group B, n = 6; 2 microgram/kg, group C, n = 7). GH concentrations in response to the single GHRH injection were comparable in the three groups. The combined administration of GHRH and CRH resulted in serum GH concentrations similar to those obtained in the same subjects in response to GHRH alone when 1 and 1.5 microgram/kg CRH were given. In contrast, the administration of 2 microgram/kg CRH together with GHRH led to an increase in GH concentrations significantly lower than those after the GHRH injection alone (GH area under the curve, 1022.18 +/- 106.26 vs. 3109.16 +/- 794.29 microgram/Lx24 h; P < 0.05). No differences in the GH response to GHRH alone or to GHRH plus CRH were detected between male and female subjects. The results of the present study indicate that CRH is capable of inhibiting GHRH-induced GH release in children. Moreover, the inhibitory effect by CRH appears to be dose dependent and not sex related.
We re-examined whether CRH stimulates GH secretion in acromegaly. Human CRH (100 micrograms) was given as an iv bolus to 15 patients with active acromegaly, and plasma GH levels were measured before and at intervals up to 120 min after the injection. For comparison, we assessed in all the patients the effects of TRH (500 micrograms), GnRH (100 micrograms), vasoactive intestinal peptide (100 micrograms) and peptide histidine methionine (100 micrograms), which are known paradoxically to stimulate GH secretion in acromegaly. A paradoxical GH response (greater than 50% above the basal) to TRH, GnRH, vasoactive intestinal peptide and peptide histidine methionine was observed in 12 (80%), 4 (27%), 5 (33%) and 2 (13%) patients, respectively. All the patients were responsive to at least one of these 4 peptides. However, none of the patients showed a positive GH response to hCRH. These results do not support a GH-releasing activity of CRH in acromegaly. Even if CRH has such an effect, it does not appear as potent as TRH, GnRH, vasoactive intestinal peptide and peptide histidine methionine. However, the possibility cannot be excluded that our negative data might have been due to the use of hCRH vs ovine CRH in earlier studies.
OBJECTIVES: A temporal association between non-rapid eye movement (NREM) sleep stages 3 and 4 and nocturnal augmentation of GH release was found long ago, yet the precise mechanism for this association has not been identified. It has been shown, however that pulsatile GHRH administration increases both slow-wave sleep (SWS) and GH. Based on these data, a role for GHRH as an inducer of SWS was proposed. To test this hypothesis, we have performed the corollary experiment whereby the action of endogenous GHRH has been antagonized. DESIGN: Healthy men (20-33 years old) had an infusion of GHRH antagonist ((N-Ac-Tyr(1), D-Arg(2)) GHRH-29 (NH(2))) or saline for a 12-h period, between 2100 and 0900 h. An i.v. bolus of GHRH was given at 0700 h and GH samples were drawn from 0700 to 0900 h to document the efficacy of GH suppression by the GHRH antagonist. METHODS: A limited montage sleep study was recorded from 2300 to 0700 h during each admission. Plasma GH concentrations were analyzed by the use of a sensitive chemiluminometric assay. RESULTS: Effectiveness of the GHRH antagonist was validated in all subjects by demonstrating 93+/-1.8% (P=0.012) suppression of GH response to a GHRH bolus. Polysomnography demonstrated that the percentage of SWS was not different when saline and GHRH antagonist nights were compared (P=0.607); other quantifiable sleep parameters were also unchanged. CONCLUSIONS: We conclude that endogenous GHRH is indispensable for the nocturnal augmentation of GH secretion, but that it is unlikely to participate in the genesis of SWS.
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OBJECTIVE: The purpose of this study was to evaluate placental growth hormone levels in maternal circulation throughout pregnancy in normal and growth hormone-deficient women with the use of a specific assay and to determine the clearance of placental growth hormone from maternal circulation after birth. STUDY DESIGN: Seventeen healthy pregnant women and 1 patient with growth hormone deficiency substituted with recombinant growth hormone during pregnancy participated in a longitudinal study from early pregnancy until birth with repetitive blood sampling and measurement of placental growth hormone levels throughout pregnancy. Furthermore, serial blood samples were drawn before, during, and after elective caesarean deliveries in 5 healthy women to calculate the half-life of placental growth hormone. Placental growth hormone was measured with the use of two monoclonal antibodies in a commercially available solid-phase iodine 125-labeled immunoradiometric assay (Biocode, Liège, Belgium). RESULTS: Placental growth hormone levels were detectable from as early as 8 weeks of gestation in some of the women and increased throughout gestation, with a maximum at approximately 35 to 36 weeks of gestation (13.7 ng/mL; range, 5.9-24.4 ng/mL) and large interindividual variations. Placental growth hormone levels did not correlate with birth weight or placental weight. In the patient with isolated growth hormone deficiency, placental growth hormone levels were detectable from 11 weeks of gestation (3.4 ng/mL) and increased throughout pregnancy to 13.9 ng/mL, which is similar to values that are obtained in the healthy pregnant women. Substitution therapy with recombinant human growth hormone did not suppress the increase in placental growth hormone. We found a mean half-life of placental growth hormone of 13.8 minutes (range, 11.5-15.2 minutes) in healthy pregnant women and an apparently similar half-life of placental growth hormone (15.8 minutes) in the growth hormone-deficient patient, assuming a monoexponential disappearance of placental growth hormone during the first 30 minutes after the delivery. After the initial 30 minutes, approximately 75% (range, 65%-89%) of the placental growth hormone had been cleared from the maternal circulation. CONCLUSION: Levels of placental growth hormone in maternal circulation increase throughout pregnancy from as early as 8 weeks of pregnancy, with maximum levels around the week 35 of gestation. The pregnancy-induced rise in placental growth hormone levels in the growth hormone-deficient patient was comparable to the rise seen during normal pregnancies and was not suppressed by the concurrent human growth hormone treatment. We speculate that maternal serum levels of placental growth hormone reflect placental function and fetal growth. However, further studies are needed to evaluate the potential clinical use of placental growth hormone determinations.
OBJECTIVE: To assess the pattern of growth hormone bioactivity (GH-BIO) and the levels of GH-binding protein (GH-BP), insulin-like growth factor I (IGF-I), and insulin-like growth factor-binding proteins (IGFBPs) in the first month of life in premature and full-term (FT) newborns. PATIENTS AND METHODS: Serum samples were collected from 9 premature newborns who were small for gestational age, 18 premature newborns who were of appropriate size for gestational age, and 20 FT newborns on the 4th and 30th days of life to evaluate the GH-BIO using the Nb2 cell bioassay, the GH levels using a radio-immunoassay (GH-RIA), and the levels of GH-BP, IGF-I and IGFBPs. RESULTS: On day 4, the GH-RIA and GH-BIO values were increased in all newborns (P < .05) compared with values in the prepubertal control subjects. The GH-BP levels were low in all newborns, with the lowest values (P < .05) found in the premature newborns and positively correlated with gestational age (P < .001). The IGF-I levels were also low, with lower values (than those found in the FT newborns) (P < .005) found in the premature group and positively correlated with the GH-BP levels (P < .001) and gestational age (P < .001). The levels of IGFBP-1 and IGFBP-2 were high, with higher values found in the premature newborns than in the FT newborns (P < .05) and negatively correlated with gestational age (P < .005). The IGFBP-3 level was lower in the premature (P < .05) than in the FT newborns and positively correlated with gestational age (P < .005). During the first month of life, the GH-RIA and GH-BIO values were significantly decreased in all newborns (P < .001), while the IGF-I level was increased in the premature newborns (P < .005). The GH-BP levels were increased only in the FT newborns (P < .001). CONCLUSIONS: The elevated bioactive GH level seen in the first few days of life seemed to be attributable to a low IGF-I level secondary to a decreased number and/or function of the GH receptors. The decrease in the serum GH level observed thereafter seemed to be secondary to an increase in the IGF-I level in the premature newborns; however, other factors may have been involved in the FT newborns in whom no increase in the IGF-I level was observed.
Using the labeled and unlabeled immunoperoxidase metods, the distribution and concentration (1) of immunoreactive LFRH-material in the hypothalamus, and (2) of gonadotropic hormones in the adenohypophysis of the ewe were determined during various reproductive stages, including two phases of the estrous cycle, anestrus, lactation, and the state after ovariectomy. The concentration of LHRH-immunoreactive material varied in particular regions of the median eminence (ME) and was closely dependent on the physiological state. The immunoreactive material was most abundant in hypothalami of lactating animals, exceeding gradually the corresponding deposits in ovariectomized animals, in ewes during the 16th day of the estrous cycle (before ovulation), and in anestrous ewes. A severe depletion of LHRH from the rostral and central parts of the ME was observed 24 h after ovulation. This depletion was accompanied by a degranulation of LH- and prolactin-producing cells in the adenophypophysis. It is postulated that LHRH is stored in the ME of the sheep in all examined endocrine stage. Only circumscribed regions of the ME take part in the cyclic release of LHRH during the ovulatory phase; this suggests a functional differentiation of the nerve terminals in this neurohemal area.
This report describes the results of the first part of the collaborative study organized by a working group sponsored by the Community Bureau of Reference of the European Community Commission. The whole study was designed to understand the causes of discrepancy among LH immunoassay methods. In the parent work, we studied the characteristics of 55 monoclonal antibodies to LH which allowed us to establish a detailed map of the antigenic surface of the hormone. In the present report we used this information to interpret the discrepancy in LH concentrations assayed with 12 different methods in 300 sera from subjects with various clinical conditions. The 55 monoclonal antibodies provided by 11 commercial companies were tested in various experiments: the apparent affinity of the antibodies was, generally but not always, higher for LH presented by a second antibody coated to plastic than for LH directly coated to plastic; 26% of the antibodies recognized the alpha subunit, 26% the beta subunit and 48% reacted only with the holomolecule (anti-alpha beta); only 28% of the antibodies were strictly specific for LH. Criss-cross experiments allowed us to distinguish 13 antigenic regions on the surface of LH: 6 were located on the alpha subunit, 3 on the beta subunit and 4 on the holomolecule. The monoclonal antibodies to the alpha beta regions further separated into 12 clusters of reactivity. Accordingly, LH appeared to exhibit at least 21 epitopes. Comparison of the immunoreactivity of various LH preparations indicated that highly purified pituitary LH and immunoaffinity purified urinary LH reacted similarly with the monoclonal antibodies and strongly differed from crude urinary LH. These data indicated that the immunoreactivity of an LH preparation depends mainly upon the degree of purification and not that much upon the origin of the preparation. The epitope specificity of the monoclonal antibodies used in 11 commercially available LH assay kits was also determined: 10 kits used at least one anti-alpha beta monoclonal antibody associated with an anti-beta monoclonal antibody in 7 cases or another anti-alpha beta monoclonal antibody in 3 cases; one kit used an anti-alpha monoclonal antibody associated with an anti-beta monoclonal antibody. None of the kits were strictly identical with regards to the epitope specificity of the monoclonal antibodies used.
In experimental animals, the decreased growth during mild uremia is not accompanied by a loss in the capacity of the pituitary gland to secrete growth hormone (GH). With the development of orally administered GH "secretagogues" (GHS), it might be possible to stimulate growth during uremia without injections. This study was designed to determine the effects of the GHS, L-163,255. Uremia was induced by 5/6 nephrectomy (NX). GHS was given orally, 3 mg/kg, twice a week. Four groups of animals included: (1) sham-operated, (2) sham-operated, pair-fed, (3) uremic (NX), and (4) uremic, GHS-treated (NX+GHS). Blood sampling was conducted via intra-atrial catheters, and GH was quantitated. Pituitary GH mRNA was measured by Northern blot, and liver GH receptor and insulin-like growth factor-I mRNAs by RNAase protection. Untreated NX animals had a specific decrease in the "mass" of the GH pulses. A burst of GH was induced by GHS, but the pulsatile pattern of GH secretion over 6 h was not affected. An increase or a return to non-uremic levels of GH-related mRNAs occurred after GHS. Thus, GHS stimulated an acute burst of GH secretion and increased specific mRNAs encoding GH-related proteins in uremic animals.