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The homeobox gene Hesx1, which encodes a pituitary transcription factor, is first expressed at gastrulation in the mouse embryo. Hesx1 expression begins in prospective forebrain tissue but later becomes restricted to Rathke's pouch, the primordium of the anterior pituitary gland. Transgenic mice lacking Hesx1 exhibit a phenotype comprising variable anterior CNS defects, such as a reduced prosencephalon, abnormalities in the corpus callosum and septum pellucidum, anophthalmia or microphthalmia, defective olfactory development and bifurcations in Rathke's pouch with pituitary dysplasia. A comparable and highly variable phenotype in humans is septo-optic dysplasia. We have cloned and sequenced the human homologue HESX1 and screened for mutations in affected individuals using single-stranded conformational polymorphism analysis. Two siblings with septo-optic dysplasia were homozygous for a missense mutation within the HESX1 homeobox. This mutation resulted in the substitution of a highly conserved arginine residue (Arg53) by cysteine and led to a loss of in vitro DNA binding. Hence, a vital role for Hesx1/HESX1 in forebrain and pituitary development in mice and humans is suggested.

Various novel growth hormone (GH) secretagogues have been developed. GH secretagogues release GH directly from the pituitary via a pathway distinct from that involving GH-releasing hormone (GHRH). However, they also act centrally to activate hypothalamic neurones, and require an intact GHRH system for potent in vivo activity. Both normal and transgenic growth-retarded (Tgr) rats release GH in response to GH secretagogues, and their responses are sensitive to the pattern of secretagogue administration. GH secretagogues are not completely specific for GH release, but also activate the adrenocorticotrophin-adrenal axis, implying that they have additional central actions. The recent cloning of an endogenous receptor for GH secretagogues now makes it possible to identify central targets for their action. An endogenous receptor implies the existence of an endogenous ligand, but its, site of production, relationship to the xenobiotic pharmacological agents and its underlying physiological relevance remain unclear. □ Growth hormone secretagogue, adrenocorticotrophin, growth hormone-releasing hormone, transgenic rats, GH secretagogue receptor, growth hormone-releasing peptide-6

Besides stimulating GH release, some GH secretagogues also release ACTH and adrenal steroids. Several novel classes of potent GH secretagogues have recently been described, and we have now tested their ability to release corticosterone in conscious normal rats. All analogs that released GH also stimulated corticosterone release to some degree, though the relative effects on GH and corticosterone varied somewhat. The corticosterone responses for some analogs were in the range of those obtained with CRF (2 μg, iv), whereas closely related analogs inactive for GH release failed to release corticosterone. Activation of the hypothalamic-pituitary-adrenal axis with GH release by GHRPs could be a highly diabetogenic combination in susceptible individuals. Therefore, a potent GHRP pentapeptide analog (G7039, 100μ g/day, sc, bid) was given to young obese male Zucker diabetic fatty rats (ZDF, n = 8/group) for 24 days. Other groups received hGH (500 μg/day, sc, bid), recombinant human insulin-like growth factor (rhI...

Human GH (hGH) acts by dimerizing two hGH receptors that bind to different sites in hGH. G120RhGH, an analog mutated in the second binding site to prevent receptor dimerization, acts as an antagonist in vitro. We have now tested the activity of this analog in vivo in rats with low or absent endogenous GH secretion. Surprisingly, treatment with G120RhGH failed to antagonize the effects of infusions or injections of hGH in hypophysectomized (Hx) rats and had little effect on hepatic GH-sensitive CYP2C transcripts in GH-deficient dwarf (dw) rats. Paradoxically, G120RhGH stimulated skeletal growth when infused into Hx rats; a pulsatile iv infusion was more effective than a continuous pattern. Coinfusion of G120RhGH with hGH produced an additive effect on growth. In addition, continuous, but not pulsatile, G120RhGH infusion elevated hepatic 2C12 messenger RNA (mRNA) expression and reduced 2C11 mRNA expression in Hx rats. The direct effects of G120RhGH on hepatic CYP2C transcripts were confirmed in cultured hepatocytes in vitro, which also revealed a significant action of PRL in elevating 2C12 mRNA expression. Binding studies revealed that G120RhGH bound preferentially to hepatic PRL receptors, as [125I]G120hGH was completely displaced by ovine PRL but was unaffected by bGH, a specific GH receptor ligand. The weak growth-promoting effects of G120RhGH were similar to those induced by recombinant hPRL in Hx rats. Our results show that G120RhGH is a poor in vivo GH antagonist in the rat, but shows a paradoxical agonist effect, probably mediated by PRL receptors in this species.

11 β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the reversible metabolism of corticosterone to inert 11-dehydrocorticosterone. At least two isoforms exist. 11β-HSD-1, the first to be characterised and the only isoform for which a cDNA has been isolated, is highly expressed in liver, kidney and hippocampus. The activity of 11β-HSD in rat liver is higher in males, due to oestrogen repression of 11β-HSD-1 gene transcription in females. Sexual dimorphism in rodent liver proteins is frequently mediated indirectly via sex-specific patterns of GH release (continuous in females, pulsatile in males). We have now investigated whether this applies to 11β-HSD, using dwarf rats (congenitally deficient in GH) and hypophysectomised animals. 11β-HSD activity and 11β-HSD-1 mRNA expression in liver was significantly lower in control female than male rats (50% and 72% of male levels respectively). These sex differences in the liver were attenuated in dwarf rats, with both males and females showing similar levels of 11 β-HSD activity to control males. Administration of continuous (female pattern) GH to dwarf male rats decreased hepatic 11β-HSD activity (30% fall) and mRNA expression (77% fall), whereas the same total daily dose of GH given in the male (pulsatile) pattern had no effect on hepatic 11 β-HSD in female dwarf rats. Continuous GH also attenuated hepatic 11 β-HSD activity (25% fall) and 11β-HSD-1 mRNA expression (82% fall) in hypophysectomised animals. However, oestradiol itself suppressed hepatic 11β-HSD activity (25% fall) and 11β-HSD-1 mRNA expression (60% fall) in hypophysectomised rats. Renal 11 β-HSD activity showed no sexual dimorphism in control or dwarf rats, although overall activity was lower in dwarf animals. By contrast, 11β-HSD-1 mRNA expression was higher in male than female kidney in both control and dwarf strains. Neither GH pattern had any effect on 11β-HSD activity or 11β-HSD-1 mRNA levels in the kidney of dwarf rats, although continuous GH attenuated 11β-HSD activity (28% fall) and 11β-HSD-1 mRNA expression in kidney (47% decrease) in hypophysectomised animals. Oestradiol attenuated renal 11β-HSD-1 mRNA expression (74% fall) in hypophysectomised rats, but increased enzyme activity (62% rise) in the kidney. None of the manipulations had any effect on hippocampal 11 β-HSD activity or gene expression. These data demonstrate the following. (i) Sexual dimorphism of hepatic 11β-HSD is mediated, in part, via sex-specific patterns of GH secretion acting on 11β-HSD-1 gene expression. (ii) There is an additional direct repressive effect of oestrogen on hepatic 11β-HSD-1. (iii) Other tissue-specific factors are involved in regulating 11β-HSD-1, as neither peripheral GH nor oestrogen have effects upon hippocampal 11β-HSD-1. (iv) The regulation of 11β-HSD-1 mRNA expression in the kidney broadly parallels the liver. The lack of correlation between changes in expression of the 11β-HSD-1 gene and renal 11β-HSD activity reflects the presence of an additional gene product(s) in the kidney, the expression of which is largely independent of GH. Journal of Endocrinology (1994) 143, 541–548

The growth hormone (GH) releasing ability of GH-releasing factor (GRF) and a GH-releasing hexapeptide, CHRP, have been studied in anaesthetized and conscious male and female rats. The GH responses to GHRP in anaesthetized rats were inconsistent, and this peptide was much less potent than GRF. Continuous iv infusions of GRF or GHRP both caused an initial GH release which was not maintained, and further GH release could be elicited by injection of GRF during an infusion of GHRP and vice versa. In contrast, conscious rats were much more sensitive to GHRP. Infusions of GHRP or GRF both caused an initial GH release. With GRF infusions, GH release continued in the normal episodic pattern whereas with GHRP infusion, GH secretion remained elevated over baseline and the normal pulsatile rhythm was disrupted. Plasma GH levels fell after stopping GHRP infusion, without an immediate resumption of normal GH pulsatility. Conscious male rats responded intermittently to injections of GRF given iv every 45 min, but when such serial injections of GRF were given during a continuous iv infusion of GHRP, the GH responses to GRF became regular and more uniform. These results suggest that GHRP prevents the normal cyclic refractoriness to GRF in male rats by disrupting cyclic somatostatin release. The greater potency of GHRP in conscious rats may also depend on the release of endogenous GRF since passive immunization with an anti-GRF serum reduced the plasma GH response to GHRP infusion. Thus in the conscious animal, GHRP may release GH by complex actions at both a hypothalamic and pituitary level.

Opioid actions on oxytocin secretion into blood and cerebrospinal fluid (CSF) were investigated in urethane-anaesthetized female rats after intracerebroventricular (icv) infusion of morphine sulphate or vehicle for 5 days. Serial femoral arterial blood samples and cisterna magna CSF samples were collected for radioimmunoassay. Naloxone was given to assess endogenous opioid tone in icv vehicle-infused rats and to precipitate withdrawal in morphine-dependent animals. Initial plasma oxytocin concentration was not affected by icv morphine infusion. In control rats receiving icv vehicle, naloxone increased plasma oxytocin 11-fold within 5 min, and in icv morphine-infused rats, naloxone increased plasma oxytocin 80-fold within 5 min. In both groups, 90 min after naloxone plasma oxytocin was still 5 and 10 times, respectively, the initial concentration. Without naloxone, neither plasma nor CSF oxytocin concentration changed significantly with time (up to 90 min) in either icv treatment group. In the icv vehicle group, there was a 2-fold increase in CSF oxytocin 90 min after naloxone. In the icv morphine-infused group, CSF oxytocin was increased 5-fold 40 min after naloxone. In another group of icv morphine-infused rats, intravenous infusion of oxytocin to achieve plasma levels similar to those seen after naloxone, did not significantly increase CSF oxytocin. In a further group of icv morphine-infused rats, [(3)H]oxytocin was infused intravenously immediately after naloxone was given; in these rats oxytocin transfer from blood to CSF could account at most for only 20% of the increase in CSF oxytocin after naloxone. A further group of rats underwent bilateral microknife ablation of the paraventricular nuclei (PVN) 9 days before icv vehicle or morphine infusions were started; blood and CSF samples were collected under urethane anaesthesia. Initial concentrations of oxytocin in CSF and in plasma were similar in both groups with PVN ablation. In all PVN-lesioned rats initial plasma concentrations of oxytocin were undetectable (

The new mutant GH-deficient dwarf (Dw) rat was used to study the effects of GH-releasing factor (GRF) or somatostatin (SRIF) on GH release. In anaesthetized adult Dw female rats, i.v. injections of GRF (0·031–2·0μg) elicited a dose-dependent release of GH. Although the peak plasma GH responses to maximal GRF doses were much lower in adult Dw rats compared with normal rats of this strain (AS), the responses largely reflected their relative pituitary GH contents (140±17 μg vs 2·9±0·4 μg, AS vs Dw (means ± s.e.m.), P < 0·001). Except at 20 days of age, normal AS rats were more sensitive to GRF than Dw rats despite their larger body weight. Peak GH responses to injection of 31·25 ng GRF increased nine-fold in normal rats between 20 and 40 days, whereas the GH responses to this GRF dose diminished in Dw rats over this age range, and their pituitary GH content was only 2–5% of that of age-matched AS rats. Treatment with human GH (200 μg/day for 7 days) stimulated growth in 40-day-old Dw rats and slightly increased the GH response to a low dose of GRF. Basal GH levels in adult Dw animals were sevenfold lower than in AS rats (2·4±0·3 vs 17·6±3·3 μg/l P < 0·001) and were further suppressed by i.v. infusion of SRIF (25 μg/h). As in normal rats, a rebound GH secretion occurred in Dw rats after stopping SRIF, which was blocked by injection of anti-GRF serum. The disappearance rate of 125I-labelled rat GH from plasma was identical in AS and Dw rats. We conclude that dwarf rats show GH deficiency as early as 20 days of age; they respond to GRF, but release only small amounts of GH due to their reduced pituitary GH content. Although basal GH release is reduced in Dw rats, the levels are higher than would be expected from the 50-fold reduction in pituitary stores, and may reflect a chronic reduction in SRIF and/or increase in GRF release induced by prolonged GH deficiency in the Dw rat. Journal of Endocrinology (1990) 127, 69–75

The pubertal development of a novel GH-deficient mutant, the dwarf rat, has been evaluated. The establishment of normal spermatogenic function within small testes suggests that GH plays no role in spermatogenic function during puberty and adult life. However, a reduction in testicular size may reflect a reduced Sertoli cell population, suggesting that GH may be of importance in prepubertal testicular development. Furthermore, marked differences between the homozygous dwarf rat and homozygous GH-deficient mouse mutants (e.g. Snell, Ames, pygmy and little mutants) have been demonstrated. It would appear that the GH deficiency in the rat mutant is far more specific for GH than those hitherto described in the mouse. In contrast to Snell dwarf mice mutants, pituitary and serum concentrations of FSH and LH are normal throughout pubertal development in the dwarf rat. Both spermatogenic function and seminal vesicle function develop normally, whilst in Snell dwarf mice spermatogenic function develops late in life and seminal vesicles remain infantile. Serum and testicular concentrations of androgen are also normal in dwarf rats. Homozygous dwarf rats have been shown to be fertile in previous studies; however, our observations suggest that despite spermatogenesis being qualitatively and quantitatively normal when assessed histologically, reduced testicular size seen in dwarf rats would lead to a reduced daily sperm output in these animals. The dwarf rat represents a mutant in which the consequences of the selective depletion of GH may be studied on various endocrine systems. The reproductive axis appears to be only partially affected, at an early age, by GH deficiency. Journal of Endocrinology (1990) 126, 193–201

Somatostatin (SS) is a powerful inhibitor of growth hormone (GH) secretion both in vitro and in vivo, and is generally regarded as having a negative influence on GH secretion and growth. In the conscious rat, the GH secretory pattern, the response to repeated injections of GH-releasing hormone (GHRH), and the feedback mechanism by which GH regulates its own release are all sexually dimorphic and probably reflect a sexually dimorphic pattern of SS release. Prolonged infusions of SS initially block GHRH-induced GH release but responses begin to break through with time. Withdrawal of SS induces a rebound release of GH in vivo, largely dependent on the release of endogenous GHRH. Prolonged exposure to intermittent infusions of SS in normal female rats produces a paradoxical growth response by inducing regular peaks of GH secretion whereas continuous infusions of SS do not. This response is GH-dependent, and is not due to other effects of SS since intermittent infusions of SS do not increase growth in dwarf rats which are deficient in pituitary GH. The sex differences in GH secretion are also reflected in the expression of several GH-dependent liver enzymes, one of which, carbonic anhydrase III, responds to manipulations of the endogenous secretory pattern by SS in both normal and dwarf rats, and appears to be sensitive to differences in basal, rather than peak, GH levels.

Carbonic anhydrase III (CAIII) occurs in male rat liver at concentrations twenty times those in the female, and is sensitive to the pattern of growth hormone (GH) release. Males release GH episodically and have high concentrations of CAIII; females produce GH in a more continuous fashion and have lower CAIII levels. In normal female rats, the endogenous GH secretory pattern was masculinized, either by regular injections of GH-releasing factor (GRF) or by intermittent infusions of somatostatin (90 min on/90 min off). Both treatments induced regular GH pulses and stimulated growth, but only intermittent somatostatin infusions raised CAIII levels (controls, 1.5 +/- 0.5; somatostatin-treated, 9.0 +/- 2.9 micrograms/mg; means +/- S.D.). GRF pulses (4 micrograms every 4 h) did not however raise CAIII levels (controls 1.8 +/- 0.5; GRF-treated 1.4 +/- 0.4 micrograms/mg). Surprisingly, hepatic CAIII is also sexually dimorphic (males, 18.8 +/- 3; females, 2.22 +/- 0.4 micrograms/mg) in a GH-deficient dwarf rat strain which has low plasma GH levels without 3-hourly GH peaks. Intermittent somatostatin infusions in female dwarf rats partially masculinized hepatic CAIII, an effect reduced by co-infusion with GRF. This CAIII response was not secondary to growth induction, since neither somatostatin nor GRF stimulated growth in dwarf rats, and pulses of exogenous GH stimulated growth in female dwarfs without masculinizing CAIII levels. Furthermore, continuous GH infusion in male dwarf rats partially feminized hepatic CAIII levels (to 9.1 +/- 2.4 micrograms/mg), whereas infusions of insulin-like growth factor-1, which induced the same body weight gain, did not affect hepatic CAIII (20.8 +/- 6 micrograms/mg). These results show that hepatic CAIII expression is highly sensitive to the endogenous GH secretory pattern, independent of growth. They also implicate the low basal GH levels between pulses, rather than the peak GH levels, as the primary determinant of the sexually dimorphic hepatic CAIII expression in the rat.

The dwarf (dw/dw) rat differs from all other rodent models of GH deficiency in that its pituitary prolactin (PRL) content is normal or even increased. We have now studied this throughout postnatal development, using a combination of immunocytochemistry, RIA and fluorescence-activated cell sorting (FACS) and analysis. Compared with normal Albino Swiss (AS) rats, adult dw/dw rats showed a profound reduction in pituitary GH content accompanied by increased PRL content, significantly so in females (AS vs dw/dw; P

Synthetic GH-releasing peptides such as GHRP-6 are potent GH secretagogues (GHSs) in several species, but attempts to stimulate growth by continuous GHS exposure have had limited success. GHSs also release ACTH and adrenal steroids. Since glucocorticoid excess is associated with poor linear growth, stimulation of the hypothalamo-pituitary-adrenal (HPA) axis by continuous GHS administration may compromise their growth-promoting effects. We have now examined the effects of continuous GHRP-6 infusion (100 mg/day, s.c. for 14 days) in normal 150-day-old female rats, and in adrenalectomized (Adx) rats with or without dexamethasone (Dex) replacement. Infusion of GHRP-6 did not significantly affect body weight gain compared with excipient-treated controls in either intact rats (controls, 9.0 +/- 1.6 vs GHRP-6, 11.8 +/- 0.9 g) or Adx rats (4.4 +/- 1.5 vs 7.9 +/- 2.7 g). However, GHRP-6 significantly increased weight gain in Adx rats treated with Dex (controls, 3.5 +/- 1.4 vs GHRP-6, 15.4 +/- 1.6 g;P

Recent studies in children suggest that there are age-related differences in the insulin-like growth factor I (IGF-I) response to malnutrition. To extend this observation, immature 4-wk-old male rats were fasted for 3 days, fed ad libitum (control), or fed 60 or 40% of control calories (restricted) and compared with 8-wk-old young adults. Over the 3-wk study period, serum total IGF-I levels of the older rats were stable despite reduced insulin levels, whereas IGF-I increased 2.2-fold in the younger controls. With the 40% diet, younger and older rats changed body weight +1 and -1 body wt/day, respectively (P < 0.0001). The restricted younger animals reduced serum IGF-I IGF binding protein-3, acid-labile subunit, and growth hormone binding protein levels significantly more than the restricted older animals. Fasting decreased most of these parameters by 40%, serum insulin by approximately 80%, and body weight by 9%, regardless of age. We conclude that the suppression of the IGF-I system in response to chronic undernutrition, but not acute fasting, is greater in maturing than young adult rats.

We have investigated the effects of different patterns of administration of recombinant human growth hormone (rhGH) on weight gain, organ growth, serum GH binding protein (GHBP) and insulin-like growth factor-l (IGF-1) levels in a series of studies using hypophysectomized (Hx) or GH-deficient dwarf (dw/dw) rats. Animals were given rhGH either by subcutaneous (s.c.) injections (1 or 2 per day) or s.c. infusions and rhlGF-1 (2 mg/kg/day) by s.c. infusion. In Hx rats, all rhGH regimes increased body weight, tibial epiphyseal plate width, and organ weights in a dose-related manner. Dwarf rats showed a smaller growth response to rhGH than Hx rats, whereas rhGH induced greater elevations in serum GHBP in drarf rats. Growth responses depended on the pattern of rhGH administration (twice daily injections > continuous infusions > daily injections). The shape of the body growth curves also differed; rhGH injections increased weight gain linearly, whereas infusions gave an initial rapid weight gain which slowed with time (a curvilinear response). For both regimens, tibial epiphyseal plate width increased linearly with rhGH dose but infusions were 5-fold more potent than daily injections. Spleen and thymus weights were markedly increased by rhGH and were also affected by the pattern of GH exposure. At 5 mg rhGH/kg/day, thymus weights were 390±35 mg for injectionsvs. 613 ± 34 mg for infusions (P

GH binding proteins (GHBPs) are present in the blood of several species and by complexing with circulating GH may alter its clearance and distribution. The actions of human GHBP (hGHBP) have hitherto been studied indirectly via their effects on the clearance of hGH. In the present experiments, recombinant preparations of hGHBP and a shorter variant lacking exon 3 (delta 3hGHBP) were tested in vivo, either alone or after preincubation with recombinant hGH, recombinant 20K methionyl-hGH, or rat GH. Multiple serial blood sampling was performed in conscious chronically cannulated guinea pigs and both GHBP and GH variants monitored in the same samples by specific RIAs, unaffected by endogenous activities in this species. hGHBP, delta 3hGHBP, hGH, and 20K met-hGH (10-40 micrograms) were all cleared rapidly when injected alone (t1/2 = 11-20 min). However, when the same amounts of hGHBP and hGH were incubated together for 60 min and then injected, the clearance of both proteins was greatly prolonged (t1/2 hGHBP = 75-94 min, hGH = 80-137 min). Similar results were obtained for hGH complexed with delta 3hGHBP, and over a range of GH:GHBP ratios from 0.3:1 to 4:1. The effect was specific for 22K hGH; neither rat GH nor 20K met-hGH altered hGHBP clearance, nor were their clearances slowed by preincubation with hGHBP. Rapid complex formation also could be demonstrated in vivo. Injections of hGH 30 min after delta 3hGHBP, or vice versa, altered the established plasma disappearance curve of hGHBP, and hGH clearance was slowed in parallel. During a continuous infusion of hGH to steady state, injections of delta 3hGHBP increased plateau hGH concentrations by forming a delta 3hGHBP/hGH complex in the circulation. These experiments show that the conscious chronically cannulated guinea pig provides a useful model in which the in vivo interaction between hGHBP and hGH can be studied dynamically. The results imply that the location, extent, and rate of complex formation between GHBP and GH may be an important determinant of the passage of GH between the intra- and extravascular compartments, which could affect the pattern of tissue exposure to GH.

Slices from ox neurohypophyses were incubated in a calcium-free medium with the ionophores A23187 or X537A. X537A (5 × 10−5 mol/l) caused a marked release of vasopressin, neurophysin and protein to the medium. A23187 (2 × 10−5 mol/l) did not cause any release by itself, but when Ca2+ was added to the medium in the presence of the ionophore, an increase in the release of vasopressin, neurophysin and protein occurred. Release of lactate dehydrogenase and peptidase were not affected by the ionophores. The secretion caused by A23187 was abolished by D600 (a verapamil analogue) (2 × 10−5 mol/l) whereas the effect of X537A was unchanged. The effects of X537A were strongly inhibited by removal of sodium from the medium. Re-addition of sodium to the medium caused a marked release. Gramicidin (10−6 or 5 × 10−5 mol/l) had no effect on secretion. Efflux of 45Ca2+ from pre-loaded slices was drastically reduced in a sodium-free medium. X537A caused an increase in the efflux rate of 45Ca2+ both in medium with a normal concentration of sodium and when slices had been incubated in a sodium-free medium. A23187 and X537A both released 45Ca2+ from a neurohypophyseal mitochondrial fraction. When sodium in a concentration of 20 mmol/l was added to this fraction, the Ca2+ accumulation was inhibited. This effect was reduced by inorganic phosphate up to a concentration of 2 mmol/l.

Brattleboro rats were housed singly in metabolic cages and continuously infused with saline. Timed urine collections were analyzed for Na+ and K+; oxytocin (OT)-like material was estimated by RIA. OT infusions reduced urine flow and increased Na+ output. Urinary Na+/K+ ratios rose during OT infusion, falling below control values after infusion. Passive immunization with an OT antiserum reduced but did not prevent these renal effects of OT. In a second experiment, immunization had no effect on daily urine volume or Na+ output, even though high titers were achieved and excretion of endogenous OT-like material was reduced. We conclude that endogenous OT has little effect on Na+ excretion in these rats, although pharmacological doses of OT produced their known natriuretic effect.

Young hypophysectomized rats were maintained with chronic indwelling i.v. cannulae attached via swivels to a multichannel pumping system programmed to deliver GH in a continuous or pulsatile pattern for several days. Continuous i.v. infusions of human GH for 5 days produced dose-dependent increases in body weight and tail length, without increasing food intake. A comparison of GH infusions by the s.c. or i.v. route showed that the direct i.v. route was threefold more effective. Pulsatile i.v. infusions of human or bovine GH at two doses (12 or 36 mu./day, eight pulses/day, 5-min duration, every 3 h) produced greater increases in body weight than continuous i.v. infusions of GH at the same daily dose. Continuous infusions of bovine GH produced a lower growth rate in the second of two consecutive 5-day treatment periods, whereas the responses to pulsatile GH did not diminish with time. Both body weight gain and long-bone growth were affected by the frequency of GH pulses; nine pulses per day were more effective than three pulses per day which in turn produced larger growth responses than one pulse per day. Keeping GH pulse frequency constant and varying pulse duration (4, 16 or 64 min) did not affect growth rates. In conclusion, long-term pulsatile i.v. infusions of GH mimic the endogenous secretory pattern, and are most effective when given at the physiologically appropriate pulse frequency. J. Endocr. (1985) 104, 53–61

The use of agarose-bound neurophysin for the extraction of oxytocin from biological fluids is described. Oxytocin can be extracted from plasma, urine and cerebrospinal fluid with a high rate of recovery and samples varying widely in volume and oxytocin concentration can be tested by the method. Columns can be used to extract and concentrate dilute samples, or to help identify small amounts of neurohypophysial hormones by affinity chromatography. The oxytocin can be eluted from the column directly into the buffer used for subsequent bioassay. The composition of the final extract is constant and independent of the composition of the sample. The specificity of the binding is high. It is suggested that the method has many advantages over others in current use.

The 'Little' mouse is characterized by a body growth rate 60% of normal due to a defect in the synthesis and storage of GH in the anterior pituitary gland. We have now investigated the effects of GH releasing factor (GRF) in these mice and in normal animals. The pituitary GH content in Little mice was only 4% of that in normal C57: +/+ mice, and was not affected by twice daily i.p. injections of human (h) GRF1-29NH2 (0·2−2 μg) for 14 days. This treatment also had no effect on body growth. In anaesthetized normal mice, single i.v. injections of 0·1 or 2 μg hGRF1-29NH2 released large amounts of GH into the plasma, whereas this peptide was ineffective in Little mice, whether or not they had been pretreated with GRF. Therefore, although pituitaries of Little mice contain significant amounts of GH, this pool is not releasable by GRF. This suggests that the dwarfism in Little mice may be partly due to a pituitary defect in GRF receptors or their stimulus-secretion coupling, rather than a deficiency in hypothalamic GRF. J. Endocr. (1985) 106, 1–5

The plasma concentration of growth hormone fluctuates widely with pronounced peaks at intervals of a few hours and troughs of nearly vanishingly low concentrations in between. The pattern of secretion varies, and different patterns affect growth differently. Tall children usually have frequent growth hormone peaks of a high amplitude, whereas short, healthy children usually have fewer peaks of a lower amplitude. Male and female rats have different patterns, and a "masculine" pattern promotes growth more than a "feminine" pattern. If the same amount of growth hormone is administered in several pulses rather than continuously, the effect on growth is much greater.

Nursing behaviour and reflex milk ejection were studied over 257 suckling periods in 26 Californian rabbits. Detailed plasma profiles of oxytocin were obtained for 161 suckling periods in 16 animals. Plasma oxytocin was detected by radioimmunoassay in serial samples of 0·2 ml blood collected during nursing. Oxytocin titres were below the lower limit of the assay (5 pmol/l) before suckling, and started to rise at a rate of 2·8 ± 0·8 (s.d.) pmol/l per s 10–30 s after the onset of suckling. Peak levels of hormone were 345 ± 113 pmol/l and were attained towards the end of nursing. In 33 of these experiments simultaneous records of activity from oxytocin neurones were taken whilst chronically sampling the blood. Each neurone gave an average of seven bursts of neurosecretory activity in suckling. Onset of this bursting pattern of discharge began 5–24 s before the rise in plasma oxytocin was detected. Oxytocin neurone activity alone was monitored in a further 55 suckling periods in eight rabbits. A marked relationship between the duration of suckling period, milk yield, peak oxytocin levels and the length of neurosecretory bursts was demonstrated over the course of lactation. All four parameters increased in parallel from day 1 to reach maximal values on days 15–20 of lactation then started to wane until the doe ceased nursing her litter on days 25–27 of lactation. J. Endocr. (1986) 108, 143–149

A new mutant GH-deficient dwarf rat has been used to study the effects of iv infusions of human GH (hGH) and recombinant human insulin-like growth factor I (hIGF-I). This animal has only about 5% of normal pituitary GH content, low circulating GH levels, and no regular GH surges. The defect seems to be specific for GH. Infusions of hIGF-I at 180 micrograms/day for 9 days elevated serum IGF-I concentrations significantly over those in the saline-infused controls (713 +/- 20 ng/ml vs. 395 +/- 31 ng/ml); hGH infusions did not raise IGF-I levels significantly (435 +/- 20 ng/ml). Gel filtration of serum samples showed that the high-dose hIGF-I infusions increased free IGF concentrations, without apparently altering the pattern of IGF-I binding whereas hGH infusions increased the amount of high mol wt IGF-I binding protein. Neither IGF-I nor hGH infusions affected the small amounts of rat GH present in the dwarf rat pituitary glands. Continuous iv infusions of hGH (200 mU/day for 9 days) stimulated body wt gain (2.1 +/- 0.2 g/day) and bone growth (96 +/- 9 microns/day) significantly compared to saline-infused dwarf rats (1.2 +/- 0.3 g/day and 43 +/- 3 microns/day). Infusions of hIGF-I at 180 micrograms/day produced a body wt gain (2.1 +/- 0.5 g/day) similar to that seen in the hGH-infused group but a significantly smaller stimulation of bone growth (63 +/- 3 microns/day). Infusion of a 5-fold lower dose of hIGF-I (36 micrograms/day for 9 days) had no effect on body wt or bone growth. Food intake was unaffected by either hGH or hIGF-I infusions. The pattern of tissue growth was affected differentially by hGH and IGF-I infusions that produced the same overall body wt gain. hGH induced a relatively proportional growth in most of the organs studied, whereas hIGF-I infusion at 180 micrograms/day stimulated a disproportionately greater growth of the kidney, adrenals, and spleen. In some of the animals, tissues were extracted for RIA of IGF-I; the amounts of IGF-I in the liver were similar in control, hGH, or IGF-I-infused animals, whereas kidney and adrenals from IGF-I infused animals contained larger amounts of immunoreactive IGF-I than did those tissues from hGH-treated rats. Thus, both hGH and hIGF-I can promote growth in the mutant dwarf rat, but they differ both quantitatively and qualitatively in their pattern of actions.

Lactating guinea-pigs were passively immunized with an antiserum to oxytocin of high titre, specificity and avidity. Single i.v. injections of 0·1–0·4 ml antiserum produced high titres which decayed slowly (half-life ≃7 days). Passively administered antiserum was effective in vivo; the clearance of exogenous oxytocin from plasma was greatly slowed in immunized animals. Passive immunization with 0·4 ml antiserum reduced milk transfer to the litter during suckling episodes of 10 min, and overall litter growth rates were significantly decreased. Non-immune serum was without effect. Plasma neurophysin levels showed the same large rises during suckling in immunized animals, indicating that neurohypophysial activation was unimpaired. Despite the presence of high titres of antiserum, some milk transfer still occurred at milk ejection. In-vitro experiments showed that more than 25% of oxytocin remained free 20 s after mixing with plasma taken from passively immunized animals. It is probable that the antiserum in the circulation was unable to bind all the oxytocin released from the posterior pituitary gland before it reached the mammary gland.

The most well established biological effects of oxytocin (OT) in female mammals are the stimulation of uterine contractions and the contraction of myoepithelial cells in the mammary gland to promote milk ejection. The physiological role of OT in milk ejection in some species is now well established, and constitutes a prime example of a neuroendocrine reflex (Cross and Harris 1952), the parts of which are now being studied in detail. The role of OT in parturition is much less clear, and varies considerably from species to species. In this brief review I shall summarize the advances that have been made in the role of OT in the milk-ejection reflex over the past few years and hopefully highlight those areas in which further studies are necessary. The role of OT in parturition will not be considered here; the reader is referred to several recent reviews in which this topic is discussed at length (Fuchs F. 1985; Fuchs A. R. 1985; Chard 1985).

The in-vivo biological activity of recombinant methionyl insulin-like growth factor I (met-IGF-I) was demonstrated in hypophysectomized rats by following blood glucose after an i.v. bolus injection of met-IGF-I; a dose-dependent decrease in blood sugar was seen. Membrane transport was studied using the non-metabolizable amino acid α-aminoisobutyric acid; stimulation was obtained with the highest dose used (90 μg/rat). To test the original somatomedin hypothesis, growth studies were performed in hypophysectomized rats. Two or three doses of met-IGF-I were given with three different administration regimes (i.v. or s.c. infusion, or s.c. injections twice daily) for 6 or 8 days. Little growth-promoting activity was observed, with a significant effect on body weight gain obtained only when met-IGF-I was given continuously at the highest dose used (180 μg/day). No effect was seen on the in-vivo uptake of radioactive sulphate into cartilage. Epiphyseal cartilage width increased slightly at the highest dose of met-IGF-I, but only when the hormone was given by infusion. When 180 μg met-IGF-I/day were given by injections, a significant effect on longitudinal bone growth was obtained (90 μm above control). The levels of IGF in the serum were not measurably increased after s.c. administration of met-IGF-I, whereas after i.v. infusion, significantly raised levels were obtained at the higher dose rates (3·0 ± 0·3 and 2·8 ± 0·1 units/ml). Growth hormone was much more effective than met-IGF-I even at 50-fold lower doses. Priming the animals with 10 mu. bovine GH/day followed by combined infusions of GH and met-IGF-I did not reveal any potentiating effects of met-IGF-I in the presence of GH. We conclude that met-IGF-I is a relatively poor growth-promoting agent when given systemically, and that somatomedins are more likely to act as local growth factors rather than as circulating mediators of the growth-promoting effects of GH. J. Endocr. (1987) 112, 123–132

The content of vasopressin, oxytocin and their related neurophysins was measured in the hypothalamus and pituitary gland of mid-trimester human fetuses. Vasopressin was present in both tissues approximately 3–4 weeks before oxytocin. The levels of the hormones in the pituitary gland increased 1000-fold over the next 3–4 months. During this time, the very high vasopressin/oxytocin ratio gradually decreased but did not reach unity in the period studied. In contrast, both the vasopressin-associated neurophysin and the oxytocin-associated neurophysin appeared in the pituitary gland at the same gestational age and showed the same exponential increase with fetal age. Lower levels of the neurophysins and the nonapeptides were found in the hypothalamus and the levels increased more slowly with fetal age. Our results suggest that the high vasopressin/oxytocin ratios observed in fetal life are due to differences in the rate of maturation of the hormone precursor, rather than to differences in the rate of de-novo synthesis.

The GH responses to single i.v. injections of GH-releasing factor (GRF) in conscious male rats are highly variable. Although normal male rats show a pulsatile secretory pattern of GH with pulses occurring at intervals of 3–3·5 h, the peaks occur at different times in individual animals. We have compared the GH responses of young conscious male and female rats to multiple i.v. injections of 1 μg human (h) GRF1-29NH2. The peak GH responses occurred 3–5 min after hGRF1-29NH2 injection and were lower in female than in male rats. Both males and females responded uniformly to hGRF1-29NH2 injections given 180 min apart and the GH responses became entrained with no endogenous GH pulsing. Female rats produced consistent GH peaks in response to hGRF1-29NH2 injections at 90-min intervals, whereas male rats responded only to alternate injections, so that GH peaks occurred only every 180 min despite giving GRF every 90 min. When the frequency of hGRF1-29NH2 administration was increased to once every 40 min female rats again responded consistently to each injection. Male rats responded intermittently, being able to respond to two injections 40 min apart, after which they became refractory to hGRF1-29NH2. This cycle of varying sensitivity to GRF in male rats probably underlies their 3-hourly endogenous GH secretory rhythm. Female rats can respond uniformly to repeated GRF injections, consistent with their more continuous pattern of endogenous GH secretion. Introducing a pulse of 10 μg rat GH into a series of hGRF1-29NH2 injections did not induce refractoriness to hGRF1-29NH2, suggesting that GH does not itself desensitize the pituitary to GRF. Whether the different patterns of GH secretion in males and females result from different patterns of GRF and/or somatostatin secretion remains to be determined. J. Endocr. (1985) 106, 281–289

Mutations in animals have provided insight into many aspects of normal and pathological human physiology. This paper reports the discovery and initial characterization of a new mutant dwarf rat. The mutation, inherited as an autosomal recessive, arose spontaneously in a breeding colony of Lewis rats at the Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford, U.K., in 1985 and the strain has now been established both in Oxford and at Mill Hill. Body growth in the mutant is retarded such that at 3 months of age both males and females weigh approximately 40% less than their normal litter-mates, and continue to grow at a slower rate. The mutants show a selective reduction in pituitary GH synthesis and storage (pituitary GH concentrations were approximately 10% of normal in males and 6% in females). The concentration of their anterior pituitary trophic hormones (LH, TSH, prolactin and ACTH) were within the normal range in dwarf animals. Exogenous GH treatment for 5 days resulted in an increase in growth rate from 1·5 ± 0·3 to 3·9 ± 0·4 g/day in male mutants, and 0·8 ± 0·2 to 3·1 ±0·1 g/day in females. Longitudinal bone growth rates were more than doubled by this treatment from 49 ± 5 to 100 ±10 μm/day in females and from 52 ± 11 to 131 ± 16 μm/day in males. Dot blot and Northern blot analysis of pituitary mRNA extracts revealed that the GH message in mutants was between 20 and 25% of normal, and that the GH transcript was of normal size. Immunoreactive GH-releasing factor (GRF) and somatostatin were present in the dwarf hypothalamus, and exogenous GRF released small amounts of GH in vivo but in proportion to the pituitary GH content. This mutant rat, which shows a selective pituitary GH deficit, may provide a useful new model for studying the endocrinology of growth. J. Endocr. (1988) 119, 51–58