Changing sensitivity of the pubertal gonadal hypothalamic feedback mechanism in man.

Clomiphene citrate in doses which stimulate gonadotropin production in the adult suppresses urinary follicle stimulating hormone (FSH) excretion and plasma testosterone concentration in prepubertal children. Such results indicate that feedback between gonad and hypothalamus is operative and highly sensitive in prepubertal humans. Puberty in man, as in the rat, is accompanied by a decrease in the sensitivity of the feedback mechanism.

The ontogenesis of human fetal hormones. III. Prolactin.

The synthesis and release of human prolactin (hPRL) in the human fetus was assessed by radioimmunoassay analysis of the content and concentration of hPRL in 82 pituitary glands and the concentration of serum hPRL in 47 fetuses of gestational age 68 days to term. Fetal hPRL exhibited parallelism with the reference standard (Lewis 203-1). hPRL was detected by 68 days of gestation (10 wk), the earliest fetal pituitary gland studied; 8 out of 33 pituitaries had a prolactin (PRL) content above 2.0 ng between 10-15 wk gestation. The mean ocntent of PRL in the pituitary gland increased sharply from 14.8 plus or minus 4.6 ng at 15-19 wk to 405 plus or minus 142 ng at 20-24 wk and 542 plus or minus ng at 25-29 wk gestation. By term, the mean content was 2,039 plus or minus 459 (range 493-3,689) and the mean concentration 15.9 plus or minus 2.4 ng/mg (range 7-20). There was a significant positive correlation (P less than 0.001) between the hPRL and human growth hormone (hGH) content of fetal pituitary glands; at term the hPRL/hGH ratio was 1/290. The concentration of serum hPRL between 12 and 24 wk ranged from 2.9 to 67 ng/ml, mean 19.5 plus or minus 2.5 ng/ml )n = 21); by 26 wk fetal serum hPRL increased sharply and attained levels of 300-500 ng/ml in late gestation. At delivery, the mean plasma concentration of hPRL was 167 plus or minus 14.2 ng/ml in 36 umbilical venous specimens and 111.8 plus or minus 12.3 ng/ml in the matched maternal venous specimens. No correlation between serum hPRL and the pituitary content or concentration of hPRL was demonstrable in 12 matched fetal specimens. In five anencephalic infants, umbilical venous hPRL levels were between 65 and 283 ng/ml. In two anencephalic infants, thyrotropin releasing factor (TRF) (200 mug IV) evoked a rise in serum hPRL in one patient from 43 to 156 ng/ml at 30 min, and in the other from 65 to 404 ng/ml at 120 min. In both patients, plasma thyroid-stimulating hormone (TSH) rose from undetectable base-line levels to peak levels of 97 and 380 muU/ml, respectively. The pattern of change in serum hPRL in the human fetus contrasts sharply with that of serum hGH, luteinizing hormone, or follicle-stimulating hormone. These observations in the fetus and in anencephalic infants suggest that the striking elevation of serum PRL in the fetus is neither mediated by a putative PRL releasing factor or by TRF, nor is a consequence of suppression or absence of PRL release inhibiting factor alone, as a functional hypothalamus is not required to attain the high PRL concentration at term. Several lines of evidence support the view that high plasma estrogen levels characteristic of gestation act directly on the fetal anterior hypophysis to stimulate PRL secretion or to sensitize the secretory mechanism of the lactotrope, increasing its responsiveness to other stimuli.

Effect of thyrotropin-releasing factor on serum thyroid-stimulating hormone. An approach to distinguishing hypothalamic from pituitary forms of idiopathic hypopituitary dwarfism.

To test the hypothesis that the primary defect in some patients with idiopathic hypopituitary dwarfism is failure to secrete hypothalamic hypophysiotropic-releasing factors, synthetic thyrotropin-releasing factor (TRF), 500 mug, wa given intravenously, and timed venous samples obtained for determination of the concentration of plasma TSH by radioimmunoassay in three groups of subjects: (a) 11 patients without evidence of endocrine or systemic disease, (group I) (b) 8 with isolated growth hormone deficiency and normal thyroid function, (group II) and (c) 9 patients with idiopathic hypopituitary dwarfism and thyroid-stimulating hormone (TSH) deficiency (group III). The mean fasting plasma TSH value was 4.1 muU/ml in group I, and 3.9 muU/ml in group II; in both groups there was a brisk rise in plasma TSH to peak levels of 12-45 muU/ml at 30-45 min, and a fall toward base line levels at 120 min. All children in group III had basal TSH levels of < 1.5 muU/ml; one failed to respond to TRF; eight exhibited a rise in plasma TSH with peak values comparable with those in groups I and II. In four of eight children in group III who responded to TRF, the TSH response was delayed and the initial rise in plasma TSH was not detectable until 10-60 min. In these four patients, plasma TSH levels continued to rise at 120 min. The mean fasting concentration of plasma thyroxine iodide (T(4)) in subjects with normal thyroid function (groups I and II) was 5.6 mug/100 ml, and the mean plasma T(4) level at 120 min was 6.6 mug/100 ml. This difference between fasting and postTRF plasma T(4) was significant (P < 0.001) by paired analysis. Mean fasting plasma T(4) concentration in group III patients was 1.3 mug/100 ml; after TRF a significant rise in T(4) concentration was not detected in this group. The results indicate that TRF test is useful in distinguishing between primary hypothalamic and pituitary forms of TSH deficiency. In light of the evidence of TRF deficiency in eight of nine patients with idiopathic hypopituitary dwarfism, it seems likely that in these patients, other pituitary hormone deficiencies may be attributable to deficiency of their respective releasing factors.

The effect of administration of human growth hormone on the plasma growth hormone, cortisol, glucose, and free fatty acid response to insulin: evidence for growth hormone autoregulation in man.

The effect of administration of human growth hormone (HGH) (3 mg every 6 hr for 6 days) on the endogenous GH response to insulin-induced hypoglycemia at 8, 12, 24, and 48 hr posttreatment was studied in 11 healthy male adults. Free fatty acid, cortisol, and glucose responses pre- and posttreatment with HGH were evaluated concurrently. Control subjects received saline injections to evaluate relationship of GH responses to the periodicity of insulin tolerance tests. The data were compared for each subject pre- and posttreatment with HGH as well as by comparison of the results of the saline-treated group with those of the HGH-treated group. The mean maximal GH concentration in response to insulin-induced hypoglycemia for all the subjects (n = 16) was 31.1 +/-3.6 ng/ml (+/-SEM) on day 1 of the control period and 23.4 +/-3.1 (SEM) on day 2, not statistically significant.A significant decrease in the maximal peak GH response (n = 8) after insulin-induced hypoglycemia was observed at 8 and 12 hr after HGH administration was terminated with mean peak values for GH of 4.6 +/-1.3 ng/ml and 10.4 +/-1.9 ng/ml, respectively (P < 0.01). A progressive return to control values was noted between 12 and 24 hr. The GH responsiveness of the saline-treated group (n = 5) was unchanged from that observed during the control period. The fasting glucose values were unchanged in the GH-treated group from those of the control period or of the saline-treated controls. Insulin resistance was apparent at 8 hr posttreatment with HGH. No differences in FFA response after insulin-induced hypoglycemia were observed in GH-treated or saline-treated subjects. The rise in plasma cortisol after insulin-induced hypoglycemia was comparable in the GH-treated and saline-treated group. Diurnal variation in plasma cortisol was maintained during the period of GH suppression. These observations support the concept that GH can modulate its secretion by means of an auto-feedback mechanism.

The ontogenesis of human fetal hormones. I. Growth hormone and insulin.

The content and concentration of immunoreactive growth hormone (GH) were measured in 117 human fetal pituitary glands from 68 days of gestation to term and in the pituitary glands of 20 children 1 month to 9 yr of age. Physicochemical and immunochemical properties of GH of fetal pituitary glands and GH from adult pituitary glands were indistinguishable by disc gel electrophoresis, immunoelectrophoresis, starch gel electrophoresis, and radioimmunoassay techniques. In the fetal pituitary gland, the GH content rose from mean levels of 0.44+/-0.2 mug at 10-14 wk of gestation, to 9.21+/-2.31 mug at 15-19 wk, to 59.38+/-11.08 mug at 20-24 wk, to 225.93+/-40.49 mug at 25-29 wk, to 577.67+/-90 mug at 30-34 wk, and to 675.17+/-112.33 mug at 35-40 wk. There was a significant positive correlation between growth hormone content of the pituitary and gestational age, crown-rump length, and the weight of the pituitary gland. The content and concentration (micrograms/milligram) of human growth hormone (HGH) in the fetal pituitary showed significant increments (P < 0.001) for each 4 wk period of gestation until 35 wk. Further increases in the HGH content were noted in pituitaries of children aged 1-9 yr (range of 832 to 11.211 mug). Immunoreactive GH was detected in fetal serum at a concentration of 14.5 ng/ml as early as 70 days gestation, the youngest fetus assayed. At 10-14 wk, the mean concentration of serum growth hormone was 65.2+/-7.6 ng/ml; at 15-19 wk 114.9+/-12.5 ng/ml; at 20-24 wk 119.3+/-19.8 ng/ml; at 25-29 wk 72.0+/-11.5 ng/ml; and 33.5+/-4.2 ng/ml at term. A significant negative correlation of serum growth hormone with advancing gestational age after 20-24 wk was observed (P < 0.001). In 17 fetuses paired serum and pituitary samples were assayed; no significant correlation between the concentration of serum GH and the pituitary content or concentration of GH was demonstrable. The serum concentration of chorionic somatomammotropin (HCS) in the fetus was unrelated to gestational age. Insulin (1-30 muU/ml) was detected in 42 of 46 fetal sera assayed. These data suggest that the appearance and development of the secretory capacity for GH by the human fetal pituitary gland coincides with developmental changes in the portal system and hypothalamus. Maturation of inhibitory central nervous system control mechanisms for secretion of GH may not occur until infancy.

Exogenous growth hormone inhibits growth hormone-releasing factor-induced growth hormone secretion in normal men.

Previous studies from this laboratory and by others in rats, monkeys, and humans support the concept that growth hormone (GH) can regulate its own secretion through an autofeedback mechanism. With the availability of human growth hormone-releasing factor (GRF), the possible existence of such a mechanism was reexplored by examining the effect of exogenous GH on the GH response induced by GRF-44-NH2 in six normal men (mean age, 32.4 yr). In all subjects the plasma GH response evoked by GRF-44-NH2 (1 microgram/kg i.v. bolus) was studied before and after 5 d of placebo (1 ml normal saline i.m. every 12 h), and then before and 12 h after 5 d of biosynthetic methionyl human GH (5 U i.m. every 12 h). The GH response to GRF (maximal increment over time 0 value) was significantly inhibited after GH treatment (0-1.3 vs. 2.3-11.2 ng/ml before treatment, P = 0.05), but was not significantly affected by placebo. This impaired pituitary response to GRF persisted for at least 24 h following exogenous GH treatment in two subjects who underwent further study. Serum somatomedin-C concentrations were significantly increased after 5 d of GH treatment (2.66-5.00 vs. 0.92-1.91 U/ml before treatment, P = less than 0.01). The impaired pituitary response to GRF may be mediated indirectly through somatomedin, somatostatin, by a direct effect of GH on the pituitary somatotropes, or by all of these mechanisms. These data suggest that after GH treatment, the blunted GH response to synthetic GRF is not solely a consequence of the inhibition of hypothalamic GRF secretion.

Secretion of unconjugated androgens and estrogens by the normal and abnormal human testis before and after human chorionic gonadotropin.

The secretion of androgens and estrogens by normal and abnormal testes was compared by determining the concentrations of dehydroepiandrosterone (DHEA), androstenedione (Delta(4)A), testosterone (T), estrone (E(1)), and 17beta-estradiol (E(2)) in peripheral and spermatic venous plasma samples from 14 normal men and 5 men with unilateral testicular atrophy. Four normal men and one patient with unilateral atrophy of the testis were given human chorionic gonadotropin (HCG) before surgery. Plasma estrogens were determined by radioimmunoassay; plasma androgens were measured by the double-isotope dilution derivative technique. Peripheral concentrations of these steroids before and after HCG were similar in both the normal men and the patients with unilateral testicular atrophy. In normal men, the mean +/-SE spermatic venous concentrations were DHEA, 73.1+/-11.7 ng/ml; Delta(4)A, 30.7+/-7.9 ng/ml; T, 751+/-114 ng/ml; E(1), 306+/-55 pg/ml; and E(2), 1298+/-216 pg/ml. Three of four subjects with unilateral testicular atrophy had greatly diminished spermatic venous levels of androgens and estrogens. HCG treatment increased the testicular secretion of DHEA and T fivefold, Delta(4)A threefold, E(1) sixfold, and E(2) eightfold in normal men. In the single subject with an atrophic testis who received HCG, the spermatic venous concentrations of androgens and estrogens were much less than in normal men similarly treated. We conclude that: (a) E(1) is secreted by the human testis, but testicular secretion of E(1) accounts for less than 5% of E(1) production in normal men; (b) HCG stimulation produces increases in spermatic venous estrogens equal to or greater than the changes in androgens, including testosterone; and (c) strikingly decreased secretion of androgen and estrogen by unilateral atrophic human tests cannot be appreciated by analyses of peripheral steroid concentrations.

Estradiol and testosterone secretion by human, simian, and canine testes, in males with hypogonadism and in male pseudohermaphrodites with the feminizing testes syndrome.

The role of the human testis in the production of 17beta-estradiol (E(2)) was investigated by determining the concentration of E(2) and testosterone in peripheral and spermatic vein plasma samples. Specimens were obtained from eight normal men, three men with hypogonadism, and two patients with the incomplete form of the feminizing testes syndrome. For comparison, similar studies were performed in four monkeys, 10 mongrel dogs, and 4 additional dogs who were given 1000 IU of human chorionic gonadotropin/day for 5 days. Plasma E(2) was measured by radioimmunoassay utilizing sheep anti-E(2) serum preceded by ether extraction and thin layer chromatographic separation of plasma steroids. Procedural blanks, which were subtracted from all reported values were 14.1+/-0.74 (SEM) pg for deionized water and 13.1+/-0.66 pg for charcoaladsorbed pooled male plasma. Pooled male and pooled female control plasmas averaged 17+/-0.71 pg/ml and 95+/-6.9 pg/ml, respectively; individual adult male specimens ranged between 8 and 28 with a mean of 18+/-1.4 pg/ml. In the eight normal men, the mean peripheral vein E(2) concentration was 20+/-1.6 pg/ml, while the spermatic vein concentration was 50 times as great, 1049+/-57 pg/ml. All three patients with testicular abnormalities had low spermatic vein E(2) concentrations (160, 280, and 416 pg/ml). Lesser E(2) gradients were found across the simian (3-fold) and canine (approximately 12-fold) testes. Testicular testosterone gradients (human 110-, simian 10-, and canine 77-fold) were greater than the E(2) gradients in all three species. In four dogs, HCG treatment elicited a 6-fold increase in peripheral and a 9-fold increase in spermatic vein testosterone concentrations; however, peripheral and spermatic vein E(2) concentrations did not differ from control values. Spermatic vein E(2) concentrations were > 4600 and 2210 pg/ml (post-HCG) in two patients with the incomplete form of the feminizing testes syndrome. Postorchiectomy, peripheral E(2) and testosterone concentrations fell precipitously in both patients, confirming the major contribution of the testes, in this syndrome, to circulating E(2) and testosterone. These studies provide direct evidence that the human testic secretes estradiol.

Failure to detect an effect of prolactin on pulmonary surfactant and adrenal steroids in fetal sheep and rabbits.

Recent reports have indicated an association between low cord prolactin (PRL) and the occurrence of respiratory distress syndrome in premature infants, and it is reported that PRL administration increases the lecithin content of fetal rabbit lung. We administered 1 mg ovine PRL to 32 rabbit fetuses on day 24 of gestation and evaluated lung phospholipid synthesis and content on day 26. Compared with diluent-injected littermates, PRL had no effect on the rate of choline incorporation into lecithin, tissue content of phospholipid and disaturated lecithin, or plasma corticoids. However, both choline incorporation and corticoids were increased in all animals undergoing surgery compared with unoperated controls. We also infused PRL (1 mg/day, i.v.) into three fetal sheep continuously over five periods of 5-8 days. Although supraphysiologic concentrations of PRL were achieved in plasma and amniotic fluid, there was no effect of this treatment on the flux of tracheal fluid surfactant or on plasma concentrations of corticoids of dehydroepiandrosterone sulfate. Thus, in this study, we failed to detect either a stimulation of the surfactant system or an adreno-corticotropic effect by PRL as previously postulated. This suggests that the relationship between PRL and respiratory distress sundrome is an indirect association.

Hormone ontogeny in the ovine fetus. XV. Studies of adenohypophysial hormones after fetal pituitary stalk section: evidence for an extrahypothalamic dopaminergic influence on fetal prolactin secretion.

The administration of a dopamine antagonist, haloperidol, to the ovine fetus in late gestation elevates plasma concentrations of PRL, suggesting tonic dopaminergic inhibition of fetal PRL secretion. The source of this dopaminergic inhibition was investigated in chronically catheterized ovine fetuses (104-135 days of gestation) after hypophysial stalk section (SS; n = 4) and in sham-operated controls (CON; n = 7). Basal PRL levels were similar in the two groups of fetuses. After the administration of TRF (250 micrograms, iv), PRL levels rose comparably in both the SS and CON fetuses. The only difference was a higher mean incremental response (P less than 0.02) in the SS fetuses. The dopamine agonist apomorphine (100 micrograms/kg, iv) induced a similar suppression of fetal PRL concentrations in CON (n = 4) and SS (n = 2) fetuses. After the administration of haloperidol (1 mg, iv) to the CON fetuses (n = 7), the concentration of fetal PRL rose (P less than 0.01). In the SS fetus (n = 4), haloperidol induced a rise in PRL concentrations (P less than 0.01); however, the response to haloperidol was less (P less than 0.01) in SS than in CON fetuses. These data suggest that there is persistent dopaminergic inhibition of PRL secretion in the fetus after complete stalk section, and that the source of this dopamine is extrahypothalamic. The greater incremental PRL response to TRH and the lesser response to haloperidol in the SS fetus than in CON are evidence for a hypothalamic component to the dopaminergic inhibition in the intact fetus. Basal FSH concentrations and the gonadotropin response to LRF were not affected by stalk section in fetuses studied 5-8 days after surgery. Both the PRL and the GH responses to 5-hydroxytryptophan were abolished by stalk section. After stalk section GH levels fell, however, significant concentrations of GH were measurable in fetal plasma in late gestation, which suggests that the fetal pituitary can secrete GH in the absence of hypothalamic stimulation at this stage in gestation.

Hormone ontogeny in the ovine fetus: XXI. The effect of insulin-like growth factor-I on plasma fetal growth hormone, insulin and glucose concentrations.

We infused intravenously recombinant human Insulin-like Growth Factor-I (IGF-I; 1 microgram/kg/min for 120 minutes after an acute dose of 25 micrograms/kg) into chronically catheterized ovine fetuses (124-132 days gestation) to study its effect on the secretion of fetal ovine Growth Hormone (oGH). In all IGF-I infused fetuses, oGH concentrations fell during the infusion. The maximal change in the concentration of oGH (mean +/- SEM) was -54 +/- 10 ng/ml in contrast to +7 +/- 6 ng/ml in saline controls (p less than 0.005), a decrease of 33 +/- 4% (controls: +6 +/- 5%; p less than 0.005). By 60 minutes after the infusion of IGF-I was completed, the concentration of plasma oGH was comparable to control and pre-infusion values. In IGF-I infused fetuses, the mean concentration of insulin also decreased (p less than 0.02), whereas glucose levels remained unaltered. The results suggest that the lack of inhibitory feedback by the relatively low levels of circulating IGF-I is one factor in the hypersecretion of GH by the fetus.

N-methyl-D-aspartate (NMDA) receptors mediate the release of gonadotropin-releasing hormone (GnRH) by NMDA in a hypothalamic GnRH neuronal cell line (GT1-1).

Previous studies demonstrated that an excitatory amino acid analog, N-methyl-D-aspartate (NMDA), stimulates GnRH secretion in the rat, prepubertal primate, and ovine fetus at the hypothalamic level. It is not known if this stimulatory effect of NMDA is mediated directly on the GnRH neurosecretory neuron. A hypothalamic GnRH neuronal cell line (GT1-1) provided a useful model system to study the effect of NMDA on GnRH release by both superfusion and static incubation techniques. Studies with GT1-1 cells indicate that GnRH neurons exhibit spontaneous autorhythmicity and function intrinsically as a neuronal oscillator for the synchronous discharge of GnRH pulses. In static incubation studies, 10(-4) and 10(-3) M NMDA increased GnRH release, whereas 10(-6), 10(-5), and 10(-2) M NMDA had no effect. A competitive NMDA receptor antagonist, AP-5 (10(-4)-10(-2) M), and a noncompetitive NMDA receptor antagonist, MK-801 (10(-12)-10(-5) M), inhibited the action of NMDA. Superfusion of GT1-1 cells after a 90-min control period followed by either continuous NMDA or intermittent NMDA (10(-4) and 10(-3) M) for 90 min increased GnRH pulse amplitude by 100-400%, but had no effect on the interpulse interval (17 min by Cluster); 10(-6), 10(-5), and 10(-2) M NMDA had no effect on either pulse amplitude or interpulse interval. MK-801 (10(-5) M) attenuated the stimulatory effect of NMDA on GnRH pulse amplitude. Incubation in glycine-free and high magnesium medium abolished the action of NMDA on GnRH release. Hybridization analysis of GT1-1 mRNA with an NMDA R1 receptor cDNA showed that this pure neuronal cell line expressed NMDA receptor transcripts observed as a 4.2-kilobase band. The results demonstrate that NMDA stimulates GnRH neurons directly to secrete GnRH through their NMDA receptors by increasing pulse amplitude without affecting pulse frequency.

Hormone ontogeny in the ovine fetus. XXIV. Porcine follicular fluid "inhibins" selectively suppress plasma follicle-stimulating hormone in the ovine fetus.

The gonads of the late gestational ovine fetus synthesize inhibin, and under the influence of FSH the concentration and content of inhibin bioactivity in fetal testes and ovaries increases. In the present study we administered inhibin-rich charcoal-treated porcine follicular fluid (pff) as a bolus to eight chronically catheterized ovine fetuses between 116 and 128 days gestation. FSH levels decreased significantly to 81.3 +/- 4.0% of baseline values after 210 min and decreased further to 71.7 +/- 4.0% throughout the sampling period (5 h). LH levels were not affected by this treatment. An estimate of the secretion rate by integration of the response curve showed a significant decrease in the concentration of plasma ovine (o) FSH after pff compared with saline injection (-28.5 +/- 10.9 U after pff vs. +19.7 +/- 8.8 U after saline; P less than 0.01), while oLH secretion remained unaltered (116.6 +/- 102.1 U after pff vs. 174.5 +/- 99.4 U after saline; P = NS). These data show that in the late gestation ovine fetus pituitary secretion of oFSH, but not oLH, is selectively decreased by inhibin-rich pff, recognizing that the net FSH-suppressing activity of pff is the sum of the actions of FSH-stimulating (e.g. activin) and -suppressing (inhibin and follistatins) factors. Thus, the inhibin-FSH feedback mechanism is potentially functional at least by 0.8d gestational age, raising the possibility of a role for inhibin in the decline of circulating fetal FSH toward term.

Hormone ontogeny in the ovine fetus and neonate. XXII. The effect of somatostatin on the growth hormone (GH) response to GH-releasing factor.

We postulated that an increase in the biological effectiveness of somatostatin (SRIF) accounts, at least in part, for the decrease in basal and GRF-induced ovine GH (oGH) secretion observed around birth in the ovine fetus and neonate. To test this hypothesis, SRIF (SRIF-14; given as 30 micrograms/kg iv bolus, followed by 2 micrograms/kg.min for 75 min) was infused into chronically catheterized fetal and neonatal lambs, and the oGH response induced by GRF [GRF-(1-44) amide; 1 microgram/kg] in the presence of exogenous SRIF was compared to the oGH response induced by GRF in saline-infused controls. In fetuses of 115-122 days gestation, SRIF had no detectable effect on the oGH response to GRF [peak incremental oGH response (mean +/- SEM), 527 +/- 124 vs. 562 +/- 103 ng/ml in controls]. In neonatal lambs (3-17 days old), SRIF completely suppressed the immediate oGH response to GRF (peak incremental response, 0.8 +/- 1.3 vs. 111 +/- 34 ng/ml in controls; P less than 0.02). In late gestational fetuses (126-139 days old), a transitional pattern was observed (peak incremental oGH response, 207 +/- 56 vs. 324 +/- 30 ng/ml in controls; P less than 0.04). In the second part of this study, we explored, in the neonatal lamb, the hypothesis that SRIF withdrawal plays a role in pulsatile GH secretion and that the amount of GRF to which the somatotrope is exposed before SRIF withdrawal is a major factor in determining the amplitude of GH bursts. SRIF (SRIF-14; a 30 micrograms/kg bolus, followed by 2 micrograms/kg.min) was infused iv for 40 min, GRF [GRF-(1-44) amide; 1 microgram/kg] was injected iv 20 min after starting the SRIF infusion, and the oGH rise after SRIF withdrawal was evaluated. In one series of controls GRF was replaced by saline, and in the other SRIF was replaced by saline. The oGH rise during recovery after SRIF alone was lower than that after the combined administration of SRIF and GRF (peak oGH increment, 8 +/- 3 vs. 38 +/- 12 ng/ml; P less than 0.04). The amplitude of the GH pulse after SRIF and GRF was similar to the immediate oGH response to GRF alone. These studies show that SRIF is unable to suppress the immediate oGH response to GRF in the ovine fetus, and that the suppressive effect of SRIF on the immediate oGH response to GRF increases gradually in late gestation and sharply at birth.(ABSTRACT TRUNCATED AT 400 WORDS)

The developmental pattern of somatostatin in the embryonic and fetal rat pancreas.

The ontogenesis of immunoreactive somatostatin in the embryonic and fetal rat pancreas has been measured by radioimmunoassay following acid extraction. Somatostatin (GIF) is detectable at 14 days gestation at a concentration of 1.6 X 10(-3) ng/pancreas. At term the content is 3.8 ng/pancreas, by 2 days neonatally, 8.3 ng/pancreas, and in the adult rat, 71 ng/pancreas through the concentration (expressed per microgram DNA) is constant from 14-19 days of gestation and reaches a level characteristic of the fully differentiated pancreas by birth. The detection of GIF in cultured pancreatic explants in the absence of innervation indicates that synthesis can occur independent of neural influence.

Hormone ontogeny in the ovine fetus: XIV. The effect of 17 beta-estradiol infusion on fetal plasma gonadotropins and prolactin and the maturation of sex steroid-dependent negative feedback.

17 beta-Estradiol (E2; 100 micrograms/kg X day) was infused iv for 6 days into three chronically catheterized ovine fetuses beginning at 105-106 days of gestation (term, 147 days) and into four younger fetuses for 3 days commencing at 87-92 days. Control studies were performed on three fetuses at each age. At 90 days, E2 had no effect on the basal concentration of either LH or FSH. In both control and E2-infused fetuses, repeated LHRH testing was associated with a decreased LH response, but the decrease was not greater in the E2-infused fetuses. The FSH response was unaffected in either group. At 105 days, E2 caused suppression of the mean basal concentration of plasma LH from 1.2 +/- 0.1 to 0.1 +/- 0.1 ng/ml (P less than 0.01) and of basal FSH from 5.6 +/- 0.5 to 3.3 +/- 0.6 ng/ml (P less than 0.05). The LH response to LHRH administration (50-micrograms iv bolus) also was suppressed by E2. The maximal incremental LH response fell from 7.1 +/- 0.4 to 3.3 +/- 0.5 ng/ml (P less than 0.01); the integrated response decreased from 5.8 +/- 0.2 to 1.8 +/- 0.2 ng/ml X h (P less than 0.01). However, no effect was observed on the rise in FSH concentration evoked by LHRH. In control studies at 105 days, basal and LHRH-stimulated gonadotropin concentrations remained constant during the experiment. These results indicate that the capacity for exogenous E2 to suppress fetal pituitary gonadotropin secretion in the ovine fetus develops between 90 and 105 days of gestation, before the normal ontogenic decrease both in the basal concentration of fetal pituitary gonadotropins and in LHRH-stimulated FSH and LH secretion, which occur later in gestation. We suggest that the development of the E2-sensitive negative feedback mechanism in the fetal hypothalamic-pituitary unit is a major factor in this decrease. At 90 days gestational age, the mean fetal plasma concentration of PRL was not affected by the infusion of E2 into the fetus (5.9 +/- 1.5 ng/ml before E2 infusion 8.1 +/- 3.2 ng/ml during E2 infusion). However, at 105 days, the mean PRL concentration rose from 49.3 +/- 18.2 to 101.6 +/- 26.1 ng/ml (P less than 0.01) after the infusion of E2. The capacity for E2 to stimulate PRL release develops in parallel with the rise in fetal plasma PRL and estrogen concentrations. The results provide evidence that circulating estrogens are a determinant of fetal PRL concentrations in late gestation.

Hormone ontogeny in the ovine fetus. XVI. Plasma immunoreactive somatomedin C/insulin-like growth factor I in the fetal and neonatal lamb and in the pregnant ewe.

The basic somatomedins [SMC/insulin-like growth factor I (IGF-I)] and the neutral somatomedins (multiplication-stimulating activity/IGF-II) exhibit different patterns during pregnancy and ontogeny. We have adapted a specific RIA for SMC/IGF-I to study the pattern of change of this growth factor in fetal, neonatal, and adult sheep plasma. Acid-ethanol extraction of the samples was performed to minimize the interference of the somatomedin-binding proteins in the RIA. Results are expressed in terms of a neonatal lamb reference plasma with an arbitrarily assigned potency of 1 U/ml (equivalent to 500 ng/ml purified human IGF-I). In chronically catheterized fetal and neonatal lambs, plasma SMC/IGF-I rose from 0.14 +/- 0.02 U/ml before 100 days of gestational age (term 147 days) to 0.22 +/- 0.01 U/ml between 101 and 144 days. In neonates (1-33 days), the mean plasma concentration of SMC/IGF-I was 0.93 +/- 0.07 U/ml. In adult sheep, plasma SMC/IGF-I was twice as high in rams as in nonpregnant ewes (0.88 +/- 0.06 vs. 0.44 +/- 0.03 U/ml); in pregnant ewes plasma SMC/IGF-I did not change significantly between the second and the last third of gestation and was always higher than the fetal levels. The rise in fetal plasma SMC/IGF-I around 100 days of gestation parallels the rise in fetal GH and PRL concentrations. The discrepancy between the present results and earlier reports based on bioassays and radioreceptor assays may be due to the presence of high circulating concentrations of IGF-II-like peptides in the ovine fetus. The striking sex difference in the plasma concentrations of SMC/IGF-I in adult sheep suggests that SMC/IGF-I generation or disposition is influenced by sex steroids.

Hormonal regulation of growth hormone secretion and messenger ribonucleic acid accumulation in cultured bovine pituitary cells.

In both primary cultures and transformed tumor cultures of rat pituitary cells, preincubation with T3 or dexamethasone increases GH secretion, mRNA accumulation, and gene transcription. GRF stimulates secretion and transcription in primary rat pituitary cultures. Sex steroids stimulate GH secretion in man, but contradictory findings have been reported in vitro. We studied the hormonal regulation of bovine GH (bGH) in primary monolayer cultures of adult bovine pituitaries. Neither T3 nor dexamethasone changed immunoreactive bGH secretion during a 3-h experimental incubation. After 72-h preincubation with T3 or dexamethasone, bGH secretion remained unchanged. T3 (10(-8) M) or dexamethasone (10(-8) M) did not alter the bGH secretory response to doses of GRF from 10(-12)-10(-8) M. However, T3 (P less than 0.001; r = 0.73) and dexamethasone (P less than 0.001; r = -0.71) decreased bGH mRNA content in dose-dependent fashions, as determined by Northern analysis and RNA dot blots probed with 32P-labeled bGH cDNA. T3 10(-7) M) decreased bGH mRNA content to 70% of the control value, 10(-7) M dexamethasone decreased bGH mRNA content to 77% of the control value, while GRF increased bGH mRNA content to 174% of the control value in a dose-dependent fashion (P less than 0.001; r = 0.72). Preincubation with testosterone or dihydrotestosterone did not change basal or GRF-stimulated GH secretion. Seventy-two-hour preincubation with 10(-8) M estradiol did not alter basal GH secretion, but increased the bGH secretory response to doses of GRF from 10(-11)-10(-8) M (P less than 0.001). Incubation with estradiol did not change bGH mRNA levels. These results demonstrate that, in contrast to rat GH mRNA, bGH mRNA accumulation is inhibited by T3 and dexamethasone. Estradiol augments the response to GRF, but this effect is not mediated by an increase in mRNA content. The hormonal responses of somatotropes vary significantly among mammalian species.

Hormone ontogeny in the ovine fetus. XVIII. The effect of an opioid antagonist on luteinizing hormone secretion.

Endogenous opioid-like peptides influence gonadotropin release in adult animals and man; however, the role of these peptides in the regulation of fetal LH secretion is not known. We administered naloxone hydrochloride (1.3 mg/kg iv), a specific opioid receptor antagonist, to 22 chronically catheterized ovine fetuses of gestational ages 94-143 days (term = 147 days). As a control, each fetus also received the vehicle on a separate occasion, the sequence of the studies being randomized. After the administration of naloxone, LH secretion increased from 38.6 +/- 5.8 to 114 +/- 21 ng/h ml-1 (P less than 0.001); LH release was not affected by administration of the vehicle. Morphine (13 mg/kg) and naloxone (1.3 mg/kg) were administered together to three fetuses (gestational age 94-105 days); LH secretion was sharply reduced from 411 +/- 14.3 ng/h ml-1 after naloxone alone to 53 +/- 17.5 ng/h ml-1 after the administration of both naloxone and morphine (P less than 0.01). The response to naloxone varied with gestational age. Fetuses of 94-115 days showed a significantly higher increment in LH secretion when given naloxone (112.3 +/- 30.7 ng/h ml-1) than did older fetuses of gestational age 126-143 days (64.8 +/- 20.8 ng/h ml-1) (P less than 0.02). These findings indicate that, in the ovine fetus endogenous opioid-like peptides exert a tonic suppressive effect on LH secretion at least as early as 94 days gestation. Moreover, the effectiveness of naloxone in augmenting LH release decreases with advancing gestational age. This latter observation supports the concept that, in the ovine fetus, endogenous opioid tone is not the sole factor involved in the dampened fetal LH secretion which is characteristic of late gestation.

Hormone ontogeny in the ovine fetus. XXVI. A sex difference in the effect of castration on the hypothalamic-pituitary gonadotropin unit in the ovine fetus.

The detection of pulsatile ovine LH (oLH) secretion in the sheep fetus by 81 days gestation (term 147 days), the suppression of fetal gonadotropin secretion by chronic administration of an LH-releasing factor agonist or antagonist, and the capacity of N-methyl; D-aspartate (a neuroexcitatory amino acid analog) to evoke a fetal oLH pulse strongly support a functional LH-releasing factor pulse-generator in the ovine fetus. In light of the sex difference in fetal gonadal function and gonadotropin secretion before day 114, we postulated that fetal castration would have a discordant effect on the pattern of gonadotropin secretion in males and females. Fetal sheep were either castrated (male = 11; female = 9) or sham operated (male = 9; female = 6) at 110-115 days gestation. Chronic indwelling arterial and venous catheters were implanted, and animals were studied for up to 30 days. During each study period fetal arterial blood samples were drawn every 15 min for 5 h and the plasma assayed for oFSH and oLH by specific RIAs. Multiple studies were performed on each fetus. In all fetuses (both intact and castrated) a decrease in oLH pulse frequency occurred after day 130. In female fetuses before day 130, castration had no effect on mean oLH pulse frequency (sham, 0.72 +/- 0.19 pulses/5 h; castrate, 0.50 +/- 0.13 pulses/5 h; P greater than 0.05). After day 130, pulsatile oLH secretion decreased in both intact and castrated female fetuses to undetectable levels during the sampling period. In contrast, castration significantly (P less than 0.001) increased mean oLH pulsatility in males before and after day 130 (less than 130 days, sham, 1.06 +/- 0.24 pulse/5 h; castrate, 2.70 +/- 0.22 pulse/5 h; greater than 130 days, sham, 0.18 +/- 0.12 pulses/5 h; castrate, 1.65 +/- 0.26 pulses/5 h). Mean oLH pulse amplitude was increased by castration only in the male fetuses (sham, 3.89 +/- 0.87 ng/ml; castrate, 6.02 +/- 0.39 ng/ml; P less than 0.05). oFSH pulses were infrequent in both sexes and not influenced by castration. The mean plasma concentration of oFSH was greater in intact female fetuses than in intact males (female, 5.65 +/- 1.15 ng/ml vs. male, 2.07 +/- 0.45 ng/ml; P less than 0.01). Castration increased the mean value for plasma oFSH in males (4.40 +/- 0.43 ng/ml; P less than 0.001) but had no effect in females (3.83 +/- 0.64 ng/ml; P greater than 0.05).(ABSTRACT TRUNCATED AT 400 WORDS)