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The mouse igf2 gene, coding for the insulin-like growth factor II (IGF-II) is parentally imprinted, only the gene copy derived from the father is expressed. To know whether IGF2, the human homologue, is also imprinted, we used an ApaI polymorphism at the 3' untranslated region in order to distinguish between mRNA derived from each copy of the gene in placentae from heterozygote human fetuses, studied after careful removal of the decidua. Six term and two pre-term placentae of heterozygotes were studied, and in each case the cDNA contained only one of the two alleles present in the genomic DNA. In three cases the mother was homozygous for the non-expressed allele, allowing assignment of paternal origin to the transcribed gene copy. We conclude that, as in the mouse, human IGF2 is parentally imprinted.

Using explant cultures of human fetal anterior pituitary glands (9-19 weeks fetal age) and an acute (3-h) test protocol, we investigated the role of two signal transduction pathways (Gs-adenylate cyclase-Gi, Ca2+ channels) in GH-releasing factor (GRF)/somatostatin (SRIF) regulation of GH secretion during the first half of gestation. Data have been analyzed for ontogenic changes using three age groups: 9-10, 12-13, and 15-19 weeks fetal age. The fetal somatotrope shows dose-related responses to forskolin (0.1-10 microM), dibutyryl cAMP (dBcAMP; 0.1-1 mM), and theophylline (0.01-1 mM), all factors that increase intracellular concentrations of cAMP; there is a significant age-related increase in the stimulatory effects of 1 mM dBcAMP and 1 mM theophylline. When any one of these three factors is added with 10 nM GRF, there are no significant additive or synergistic effects on GH secretion. Although 10 nM SRIF has no effect at 9-10 weeks, it is inhibitory in the 12-13 and 15-19 week groups, significantly suppressing the stimulatory effect of 1 microM forskolin and completely blocking the effects of 1 mM dBcAMP or theophylline. Pretreatment of cultures with pertussis toxin completely blocks SRIF inhibition of both basal and GRF-stimulated GH release. KCl (5-50 mM) and Bay K 8644 (0.1-10 microM), both activators of Ca2+ channels, have dose-related stimulatory effects on GH release; 50 mM KCl shows an age-related increase in stimulatory activity. If 10 nM GRF, 1 microM forskolin, 1 mM theophylline or 1 mM dBcAMP is added with either 50 mM KCl or 1 microM Bay K 8644, there is an additive response. SRIF (10 nM) completely blocks the stimulatory effects of 1 microM Bay K 8644 and markedly inhibits the effects of 50 mM KCl from as early as the ninth week of fetal age. The Ca2+ channel blocker nifedipine (1-10 microM) significantly inhibits basal as well as stimulated (GRF, forskolin, dBcAMP, theophylline, KCl, and Bay K) GH release from as early as 9 weeks fetal age; in contrast, the calmodulin blocker trifluoperazine (0.01-10 microM) has no effects on basal GH secretion and only slightly inhibits the stimulatory effects of KCl. Pretreatment with 10 nM GRF for 24 h significantly decreases a subsequent 3-h response to 10 nM GRF, but does not alter the subsequent response to 1 mM theophylline or 50 mM KCl.(ABSTRACT TRUNCATED AT 400 WORDS)

We have previously shown that gamma-interferon promoted the proliferation of adult human astrocytes isolated from brain biopsy specimens. In contrast, in the present study, astrocytes derived from neonatal mouse brains and treated with recombinant murine gamma-interferon responded by a decrease (average of 50% at 100 U/ml) in proliferation. The basal rate of proliferation as assessed by bromodeoxyuridine incorporation was markedly increased in neonatal mouse astrocytes when compared to the adult human cells, suggesting that age, and the corresponding metabolic activity of cells, could be important determinants in the mitogenic response of astrocytes to cytokines. However, subsequent examinations of fetal human and adult mouse astrocytes, with comparable basal rate of proliferation to neonatal mouse and adult human cells respectively, showed gamma-interferon to promote DNA synthesis in fetal human astrocytes while inhibiting that of adult mouse astrocytes. The results suggest species differences in the proliferative response of human and mouse astrocytes to the cytokine gamma-interferon.

There is now strong evidence to suggest that insulin-like growth factor I (IGF-I) plays an important role in breast cancer proliferation. Recently we observed that tamoxifen-treated stage I breast cancer patients have serum IGF-I levels significantly lower than placebo-treated patients. Since IGF-I is growth hormone (GH) dependent, we have tested the hypothesis that tamoxifen alters serum IGF-I levels through direct inhibition of GH secretion. Immature lamb pituitary cultures were examined for acute (3 h) or chronic (1-6 day) effects of the drug, using doses (0.1-10 mumol/l) based on known steady state levels in patients on tamoxifen therapy (0.31-3.1 mumol/l). Tamoxifen had a direct, dose-related, inhibitory effect on GH release from pituitary somatotropes, during acute as well as chronic treatment. The 10 mumol/l dose consistently decreased both basal and growth hormone releasing factor stimulated GH release. These in vitro data are consistent with our hypothesis that tamoxifen suppresses serum IGF-I levels by acting at the pituitary to inhibit GH release.

Epidermal growth factor exerts potent receptor-mediated mitogenic effects on a variety of target cells in vitro, but its importance in normal organ development is not yet fully understood. We report that the specific high-affinity receptors for EGF/TGF-Alpha increase dramatically in late gestational rat kidney (from 2.3% at 16 days gestation to 6.4% at term) and then fall toward basal adult levels (< 1% binding) during the first week of post-natal life. This post-natal fall-off in EGF binding corresponds temporally to the period when replication of rat kidney DNA begins to slow (4-7 days of post-natal life). EGF mRNA is not detectable in rat kidney by Northern analysis until the second week of post-natal life, but high levels of transforming growth factor-alpha are demonstrable by specific radioimmunoassay in extracts of fetal kidney (52.2 +/- 8.2 pmoles/gram kidney) and amniotic fluid (4.49 +/- 0.75 pmoles/ml). We speculate that induction of EGF-receptors in fetal rat kidney may confer responsiveness to local transforming growth factor-alpha and dictate the rate of hyperplastic renal growth in the perinatal period.

By the end of gestation, nephron formation in the human kidney is complete. Following local induction of metanephric mesenchyma, committed cells of each primitive renal vesicle must undergo a phase of rapid cell division. In order to identify genes which might regulate these events, we examined the expression profile of 22 proto-oncogenes in fetal versus adult human kidney. Among those expressed at especially high levels in the fetal tissue was the gene for epidermal growth factor receptor (EGFR). We were able to detect mRNA (by polymerase chain reaction [PCR] amplification) and peptide (by specific radioimmunoassay) for transforming growth factor-alpha (TGF-alpha) in fetal kidney, whereas epidermal growth factor (EGF) peptide was undetectable in midgestation kidney and amniotic fluid. TGF-alpha/EGFR interactions may direct renal cell proliferation in fetal life.

Beginning at the fifth week of fetal life, successive generations of individual nephrons are induced by contact between metanephric mesenchyme and ureteric bud. Following phenotypic transformation, cells of each primitive renal vesicle undergo a phase of rapid cell division. In order to identify genes which might regulate nephron development in man, we screened adult and fetal kidney RNA for expression of a panel of growth-related genes. Among the genes which were expressed at higher levels in fetal kidney was the epidermal growth factor (EGF) receptor. There is controversy as to the most likely physiologic EGF receptor ligand in fetal kidney; we were able to identify a transcript for transforming growth factor-alpha (TGF-alpha) but not EGF on Northern blots of fetal kidney RNA. Since the abundance of TGF-alpha mRNA is low, we confirmed its presence by polymerase chain reaction amplification. Using specific radioimmunoassays, we also provide direct evidence for TGF-alpha but not EGF peptide in extracts of fetal kidney and mid-gestational amniotic fluid. We suggest that TGF-alpha/EGF receptor interactions may serve an important function in development of human fetal kidney.

To examine the effects of anabolic agents given during late gestation on the maternal and fetal somatotropic axes, we injected pregnant ewes twice daily with 0.15 mg somatocrinin (GRF)-(1-29) for 10 days beginning on day 130 of gestation. Maternal and fetal endocrine changes were compared with control animals using both in vivo and in vitro approaches. Treatment with GRF increased maternal plasma levels of growth hormone (GH) and insulin-like growth factor I (IGF-I;P less than 0.05) but not IGF-II. Under in vitro test conditions, maternal pituitary cells showed a greater maximal response (P less than 0.001) to GRF. In the fetuses of treated ewes, cord plasma GH levels were not significantly increased compared with controls. These animals had similar IGF-I but higher IGF-II (P less than 0.05) plasma levels. The maximal response of fetal pituitary cells to GRF was increased (P less than 0.001). GRF treatment had no influence on maternal and fetal pituitary cell responses to somatostatin under either basal or GRF-stimulated conditions. In addition, these treatments did not affect plasma levels of placental lactogen, glucose, or free fatty acids in the maternal and fetal sheep. These data are compatible with the hypothesis that treatment of pregnant ewes in the last days of gestation with GRF could support accelerated fetal growth.

We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10(-6) M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differentiated function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.

In Henoch-Schönlein purpura nephritis (HSPN), glomeruli may develop cellular "crescents" composed of infiltrating monocytes and proliferating renal epithelia. In this study, we demonstrate that peripheral human monocytes can release an epidermal growth factor (EGF)-like substance detectable by a radioreceptor assay, which recognizes both EGF and transforming growth factor-alpha (TGF-alpha), but not with a radioimmunoassay, which recognizes only EGF. Furthermore, we report that urine from pediatric patients during the acute phase of HSPN contains a similar EGF-like species in addition to the endogenous EGF which is normally present. The EGF-like material was not present in urine from nine healthy children or from six children with acute post-streptococcal glomerulonephritis. The extent of crescent formation in our patients is uncertain, since renal biopsy was performed in only one case. However, we speculate that the urinary material resembling TGF-alpha which appears during the acute phase of HSPN may derive from monocytes infiltrating the kidney.

The ability of human adult adrenal to utilize progesterone (P4) for cortisol (F) synthesis in vitro has been compared with that of fetal adrenal tissue. Explant cultures were studied for 6 days using Ham's F10 medium supplemented with 10% fetal bovine serum (FBS), with or without added P4 as substrate. Short-term (4h) incubations of fresh tissue minces were carried out in Ham's F10, with or without P4, in the absence of FBS. In contrast with the fetal gland, F production by cultured adult tissue was unaffected by addition of P4. During short-term incubations, P4 increased F production 150-fold in the fetal tissue as compared to 2- to 4-fold in the adult. ACTH had no acute effect on the P4 to F conversion in either tissue. These results demonstrate that the fetal adrenal exhibits a greater ability to utilize P4 for F production than the adult adrenal.

Conditioned medium from human placental monolayer cultures (PM) had a marked stimulatory effect on proliferation (3H-thymidine uptake) of human fetal zone adrenal cells in primary monolayer culture, even in the absence of serum. Epidermal growth factor (EGF) and fibroblast growth factor (FGF) also significantly stimulated fetal adrenal cell growth. However, the effects of PM differed from those of EGF and FGF in several respects: 1) maximal response to PM was 2-5 times greater; 2) mitogenic effects of EGF and FGF were suppressed by adrenocorticotropic hormone (ACTH), whereas that of 50% PM was not; 3) PM inhibited ACTH-stimulated steroidogenesis (dehydroepiandrosterone sulfate and cortisol), but EGF and FGF did not. Preliminary characterization studies have indicated that approximately half of the placental growth-promoting activity is heat resistant and sensitive to bacterial proteases, and that 50-60% of the activity is lost after dialysis with membranes having a molecular weight cutoff of 3500. These findings suggest a role for the placenta in the growth and differentiated function of the human fetal adrenal gland.

Studies were undertaken to determine whether the insulin-like growth factors (IGF-I and -II), bind to specific membrane receptors in the pituitary and brain. Anterior pituitary glands, hypothalami, and brains (minus hypothalami) were obtained from adult male Sprague-Dawley rats (225-300 g) and 15,000 X g membranes prepared by differential centrifugation. Binding of 125I-IGF-I and 125I-IGF-II to all three membrane preparations was specific, time and temperature dependent, reversible, and increased in proportion to increasing concentrations of membrane protein or labeled ligand. Neither the pH of the assay buffer (6.5-8.5) nor the presence or absence of 1 mg/ml bacitracin had any significant effect on the levels of specific binding. In all three membrane preparations IGF-II specific binding was 3-5 times higher than that observed for IGF-I, and unlabeled IGF-II displaced either 125I-IGF-I or 125I-IGF-II better than comparable concentrations of IGF-I. All three membrane preparations showed similar low specific binding of 125I-insulin (1.3-2.2%) and negligible specific binding of 125I-rat GH (less than 0.5%). The presence of specific IGF and insulin receptors in rat anterior pituitary, hypothalamic, and brain tissue is additional evidence that IGFs and insulin are involved in modulating brain and pituitary function.

The total number and saturation of nuclear receptors for T3 were measured in human fetal brain and lung from the 9th to the 13th week of fetal life. The concentrations of occupied and unoccupied receptor sites were determined by measuring total binding capacity at 0 and 22 C. At 0 C [125I]T3 was bound mainly to unoccupied sites, whereas at 22 C it was bound to unoccupied sites plus a fraction (70%) of endogenously occupied sites. Saturations of brain and lung receptors were similar (12-27%). From the fractional receptor occupancy and the receptor dissociation constants (34 pmol/L in brain and 56 pmol/L in lung) the concentration of intranuclear free T3 was calculated to be 9 pmol/L in brain and 11 pmol/L in lung. Total and free cytosolic T3 were measured by RIA and equilibrium dialysis. Total T3 was below the limit of detection in lung (90 pmol/L). The concentration of free T3 in brain cytosol was 0.95 pmol/L at 11 weeks and 2.96 pmol/L at 13 weeks, i.e. considerably lower than the nuclear free T3 concentration. These results suggest the presence of a small gradient (3-fold) between nuclear and cytosolic free T3 in both fetal tissues. The data strongly support the idea that thyroid hormones influence human brain development at least from the 9th to the 10th week of gestational age.

Adrenals obtained from human abortices at midpregnancy were kept under conditions of tissue culture and the production of cortisol and dehydroepiandrosterone sulfate (3beta-hydroxy-5-androsten-17-one, 3-sulfate; DHA-S) monitored by radioimmunoassays for up to 2 weeks. Basal production of dehydroepiandrosterone sulfate was considerably higher than that of cortisol. alpha1-24corticotrophin, alpha1-39corticotrophin, (a long acting porcine corticotrophin) at the concentration of 1 mU/ml of culture medium in both instances, and dibutyryl cyclic AMP (1mM) enhanced the production of both steroids some 3 to 30 times above control values. Medium harvested from homologous pituitary cultures had comparable corticotrophic activity. It is concluded that at midpregnancy the regulation of corticoidogenesis implies the existence of a corticotrophic factor either identical or closely related to ACTH.

Using an explant culture system, we have demonstrated that human somatotropes respond to growth hormone releasing factor (GRF) and somatostatin (SRIF) from as early as 9.5 weeks of fetal age. Responsiveness to GRF increases significantly as a function of age up to midgestation while SRIF inhibition of basal growth hormone (GH) release does not change. SRIF has little effect on GRF-stimulated GH secretion from early gestation pituitaries, but its ability to block GRF stimulation gradually increases with fetal age from 9.5 to 16 weeks. The response to GRF remains predominant throughout this developmental period: 100 times more SRIF than GRF must be added to the cultures in order to block the GRF stimulatory effect and maintain GH secretion at basal (control) levels. Finally, adding SRIF 30 min prior to the GRF does not increase the inhibitory activity of SRIF. Our data suggest that the mechanisms that permit an interaction between GRF and SRIF are developing, but slowly, in the early to midgestation human fetal somatotrope and that GRF stimulatory pathways predominate. This may help to explain the very high levels of GH in fetal serum during the first half of gestation.

Human placentae of different gestational ages have been used to investigate the binding and degradation of insulin as well as the regulation of insulin membrane receptors. Bacitracin was found to be an effective inhibitor of insulin degradation in human early gestation and term placental cell cultures. In the presence of bacitracin, [125I]-insulin bound rapidly and reversibly: maximal binding occurred at 4 degrees C, with a sharp pH optimum at 7.5, and exhibited a high degree of specificity. The extent of binding was proportional to cell protein and [125I]-insulin concentrations. Term (38 to 41 weeks; n = 25) placental cell cultures possessed receptors for insulin that were increased three-fold compared to early gestation (8 to 18 weeks; n = 17). This was due to an increase in receptor number with no significant alteration in affinity. A decrease in insulin binding in both early gestation and term placental cells was related to both the insulin and bacitracin concentrations present during 12 to 20 h of preincubation at 37 degrees C. The receptor loss was due to a decrease in the number of receptors per mg cell protein with no apparent change in their affinity. We conclude that our in vitro system, which utilizes human placental cells in monolayer culture, will permit a more direct study of the metabolic effects of insulin in both early gestation and term placentae.

This study examined the effects of human chorionic gonadotropin, adrenocorticotropin, human luteinizing hormone, and mouse epidermal growth factor on growth (thymidine incorporation) and steroidogenesis (dehydroepiandrosterone sulfate production) of human fetal zone adrenal cells in monolayer culture. Two preparations of human chorionic gonadotropin extracted from pregnancy urine were used, one highly purified (National Institutes of Health, CR-121) and one less pure (Sigma). Thymidine incorporation was increased twofold to tenfold in cultures exposed to the Sigma human chorionic gonadotropin preparation or to mouse epidermal growth factor as compared to control. Pure National Institutes of Health human chorionic gonadotropin and luteinizing hormone had no effect on growth. When adrenocorticotropin was added alone or in combination with Sigma human chorionic gonadotropin or mouse epidermal growth factor, growth was decreased. Dehydroepiandrosterone sulfate production was stimulated by adrenocorticotropin but not by human luteinizing hormone, human chorionic gonadotropin, or mouse epidermal growth factor. These results suggest that human pregnancy urine contains a growth factor which remains to be identified but that pure human chorionic gonadotropin has no mitogenic or steroidogenic effects on the cultured fetal zone cells of the human fetal adrenal gland.

We have examined the effect of the catechol oestrogens 2-hydroxyoestradiol (2-OHE2), 4-hydroxyoestradiol (4-OHE2) and 2-hydroxyoestrone (2-OHE1) and their corresponding primary oestrogens on secretion of LH and FSH by enzymatically dispersed rat anterior pituitary cells in monolayer culture. Basal LH levels in the medium were significantly higher than in control wells when cells were exposed to 10(-8) M-oestradiol-17 beta for 40 h: oestrone and all three catechol oestrogens (in the same doses) also stimulated basal LH concentrations to levels quantitatively similar to those seem after oestradiol treatment. The same effects were observed when steroids were given at 10(-9) mol/l. Oestradiol, 2-OHE2, and 4-OHE2 but not 2-OHE1 increased pituitary responsiveness to LH releasing hormone (LH-RH) (given in a range of doses from 10(-11) to 10(-6) mol/l). The responses of cells treated with 2-OHE2 and 4-OHE2 were similar, though less than the response seen after treatment with oestradiol. This contrasts with the very different oestrogenic effects of 2- and 4-OHE2 previously observed in vivo. Neither oestradiol nor the catechol oestrogens had any effect on basal or LH-RH-stimulated FSH release.

We have reevaluated IGF binding specificity to membrane receptors in rabbit mammary gland (RMG) and hypophysectomized rat liver (HRL) using recombinant DNA-derived and synthetic analogues of human IGF-I and highly purified IGF-II. SDS-PAGE demonstrated that [125I]IGF-I bound to type-I IGF receptors in RMG; this binding was inhibited in a similar fashion by the IGF-I analogues (IC50 = 10 ng/ml) and to a lesser extent by IGF-II (IC50 = 60 ng/ml). [125I]IGF-II bound to type-II IGF receptors in both RMG and HRL. The IC50 for IGF-II was 9 and 3 ng/ml with RMG and HRL, respectively. At a dose as high as 1 microgram/ml, IGF-I analogues inhibited less than 20% of [125I]IGF-II binding. These results suggest that IGF-I has little or no affinity for type-II IGF receptors.

Explants prepared from the neocortex and the fetal zone of the human fetal adrenal (gestational age 13 to 18weeks) were maintained under conditions of organ culture for 7 to 9 days during which time they were exposed to hACTH and various related peptides. Corticotrophic activity was monitored by the daily release of dehydroepiandrosterone sulfate (3beta-hydroxy-5-androsten-17-one, 3-sulfate; DHA-S) and cortisol as quantified by radioimmunoassay, hACTH (2.2 x 10(-9) - 2.2 x 10(-8)M) was the most active in sustaining steroidogenesis by both neocortical and fetocortical cells. alpha-MSH possessed similar properties but not at concentrations lower than 10(-6)M, whereas CLIP (4.4 x 10(-9) - 1.1 x 10(-7)M), the 18-39 C-terminal moiety of ACTH, was devoid of activity. Corticotrophic activity with respect to fetocortical explants appeared to be that of maintenance of function best illustrated by dehydroepiandrosterone sulfate biosynthesis, while enhancement of steroidogenesis was observed in the neocortex as manifested by cortisol release. Although not eliminating the possible existence of a specific fetal corticotrophin related to ACTH1-39, the data indicate that hACTH is capable of regulating steroidogenesis in the fetal zone which is primarily geared to the formation of dehydroepiandrosterone sulfate.

Using a monolayer approach, we have examined the acute (3 h) effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on GH release from pituitary cells of male and female 70-, 100-, and 130-day-old fetuses and newborn lambs and of prepubertal male lambs. GRF stimulated basal GH release in a dose-dependent (10(-12)-10(-8) M) manner at each stage in development. There was no linear relationship between maximal response and increasing age of the donor animals. The ED50 values for GRF were similar in all groups, except in the pituitaries from male and female 130-day-old fetuses, where the ED50 values were significantly higher. SRIF elicited a dose-related (10(-10)-10(-6) M) inhibition of basal GH secretion at each stage of fetal life and in the prepubertal period; although the response was lower in the youngest fetal pituitaries, there was no significant change in maximal response during the fetal or prepubertal period. No effect of SRIF on basal GH secretion was observed in newborn lambs. However, SRIF (10(-7) M) was able to block GRF (10(-8) M)-stimulated GH release in 100- and 130-day-old fetal and prepubertal as well as newborn lamb pituitary cells. Plasma IGF-I concentrations increased from 15.0 +/- 0.7 (mean +/- SE) and 13.8 +/- 0.9 ng/ml for male and female animals, respectively, at 70 days gestation to 55.8 +/- 3.2 and 51.8 +/- 11.1 ng/ml at the time of birth. The increase was much more pronounced in prepubertal lambs, especially in male animals, where IGF-I levels reached 300.8 +/- 37.7 ng/ml. IGF-I (100 ng/ml) had no effect on basal GH release in 70- and 100-day-old fetal, newborn, and prepubertal lamb pituitary cultures, but significantly inhibited basal GH secretion from 130-day-old fetal cells. This dose of IGF-I had no effect on GRF (10(-9) M)-stimulated GH release at 70 days gestation. It significantly inhibited this effect at 100 days and in prepubertal lamb cells. In 130-day-old fetal and newborn lamb pituitary cultures, IGF-I completely blocked the GH response to GRF.(ABSTRACT TRUNCATED AT 400 WORDS)

Unlabelled: To determine the direct effects of insulin-like growth factors (IGFs) on pituitary secretion of GH, PRL, and ACTH, adult male rat pituitary explant cultures were tested with acute (3-4 h) or chronic (24 h) exposure to a semipurified preparation of IGF peptides, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. To examine the effect of serum binding proteins on IGF bioactivity, certain experiments were run in parallel using culture medium supplemented with 10% fetal bovine serum or 1% purified BSA. To compare IGF effects with those of known regulators of pituitary function, cultures were also tested with SRIF, TRH, human pancreatic GH-releasing factor, insulin, and human GH (hGH). IGFs, at 10-100 ngeq/ml, were able to inhibit significantly both basal and (1 mM) theophylline-stimulated rat GH (rGH) and rat PRL (rPRL) release during acute (3-4 h) exposure. Only the higher concentration (100 ngeq/ml) was consistently effective in inhibiting rGH and rPRL output after 24 h in culture, due to gradual metabolism of IGF peptides by the cells. Parallel experiments carried out in medium containing 10% fetal bovine serum or 1% BSA gave similar results, demonstrating that IGF serum binding proteins did not interfere with IGF bioactivity in this test system. Chronic 5-day exposure to IGFs, at 100 ngeq/ml, resulted in a significant inhibition of rGH release for the entire 5-day period and rPRL release for the first 3 days. IGFs (10-100 ngeq/ml) had no acute or chronic effect on basal or theophylline-stimulated ACTH release. Purified IGF-I (50 ng/ml) and IGF-II (50 ng/ml) gave approximately equivalent effects on basal rGH and rPRL release during an acute (3 h) exposure suggesting that both IGFs can exert inhibitory influence on pituitary function. Ten thousand nanograms per ml insulin and 10(-9) M SRIF had acute inhibitory effects on rGH and rPRL release similar to what were observed for 100 ngeq/ml semipurified IGFs. hGH (200 and 1000 ng/ml) had no effect on rGH, rPRL, or ACTH release when administered either acutely (3-4 h) or chronically (24 h)., Conclusions: These studies demonstrate that IGFs, administered acutely or chronically, directly inhibit basal as well as theophylline-stimulated rGH and rPRL output by the rat pituitary; ACTH release remains unaltered. Insulin, at high concentrations, can mimic these effects, whereas hGH has no effect either acutely or chronically.

We have examined the regulation of GH secretion from monolayer cultures of prepubertal male lamb anterior pituitary cells. Growth hormone-releasing factor (GRF 1-44) stimulated GH release in a dose-related manner: the maximal effective dose was 10(-10) M, which caused a 500% increase in basal GH secretion, while the half-maximal effect was reached with a dose of 2.5 x 10(-11) M (ED50). Thyrotropin-releasing hormone (TRH) also elicited a dose-dependent stimulation of GH secretion, although it was approximately 1000 times less potent than GRF. GRF and TRH did not have additive or synergistic effects on GH secretion. Somatostatin (SRIF) at a concentration of 10(-7) M maximally inhibited basal GH release to 40% of that of the control; the ED50 was 2.0 x 10(-9) M. Moreover, 10(-7) M SRIF blocked the stimulation of GH secretion induced by 10(-8) M GRF. However, when the cells were incubated with these two peptides at an identical concentration (10(-8) M), GH secretion was stimulated significantly above control values. When added at the same concentration (10(-7) M, TRH ans SRIF nullified their respective effects. A dose of 100 ng/ml of synthetic IGF-I was without effect on basal GH release, but significantly decreased 10(-9) M GRF-induced stimulation of GH secretion. these data indicate that in prepubertal male lambs: the stimulatory effect of GRF is predominant over the inhibitory effect of SRIF, somatostatin inhibits TRH stimulation of GH secretion in vitro, and IGF-I may control GH secretion by modulating GRF effects at the pituitary level.

Human foetal adrenal cells were grown in monolayer culture and their steroidogenic capacity observed for up to a month. The cells produced a complex array of steroids and some of their ester sulphates from endogenous as well as from [14C] and [3H] precursors. ACTH stimulated corticoidogenesis, particularly cortisol secretion, and markedly enhanced the incorporation of progesterone and pregnenolone into cortisol. Following incubation with the same precursors, large amounts of radioactivity remained water soluble. From the butanol extractable material of this fraction, dehydroepiandrosterone sulphate was characterized as the main metabolite of pregnenolone and corticosterone and 11-deoxycorticosterone sulphates as the main metabolites of progesterone. With time in culture there was a decrease in steroidogenesis as well as a steady decline in responsiveness to ACTH, mainly manifested by cortisol secretion. The medium from homologous foetal pituitary cultures stimulated cortisol production by the human adrenal cell monolayer.

Studies of human fetal adrenal function and its control have revolved mainly around the remarkable capacity of the unique fetal zone of this gland to elaborate dehydroepiandrosterone sulfate. Another important function of the fetal adrenal, however, is its production of cortisol. Because the human fetal adrenal is deficient in 3 beta-hydroxysteroid dehydrogenase activity, cortisol has been thought to be formed from circulating progesterone. To further investigate this hypothesis, cortisol production by separated fetal and definitive zones of the midgestation human fetal adrenal in organ culture has been examined in the absence and presence of varying concentrations of progesterone and adrenocorticotropic hormone. Cortisol was measured by radioimmunoassay. In the absence of progesterone, cortisol production by both zones increased gradually over time in culture in response to adrenocorticotropic hormone. In the presence of progesterone, cortisol production by the definitive zone was unchanged. In contrast, the response of the fetal zone to progesterone was immediate: cortisol production increased significantly and remained high throughout the culture period. These results suggest a greater capacity of the fetal zone to utilize progesterone for cortisol production and are consistent with morphologic evidence that the active zone of the midgestation human fetal adrenal is the fetal zone, possessing not only the enzyme activity necessary for dehydroepiandrosterone sulfate production but, except for 3 beta-hydroxysteroid dehydrogenase, that for cortisol production as well.

To determine the direct effects of insulin-like growth factors (IGFs) on hormone release by the human pituitary gland, human foetal, adult normal and tumour pituitary tissues were maintained in culture for 2 to 4 weeks and tested with acute (3 h) exposures to different preparations of IGF peptides. Adult normal pituitaries and adenomas were tested with a semipurified preparation of IGFs, free of immunoreactive insulin, containing IGF-I and IGF-II in a ratio of approximately 1:4. Human foetal pituitaries were tested with the semipurified IGFs as well as more purified preparations of IGF-I and IGF-II. Culture media were assayed for hGH, hPrl, hACTH and hLH using specific radioimmunoassays. Both foetal (n = 16 (No. of pituitaries), 33 (No. of observations] and normal adult (n = 3, 16) human pituitaries cultures responded to the semipurified IGFs (2-25 ngEq/ml for foetal and 2-4 ngEq/ml for adult pituitaries) with a significant decrease in hGH release compared to basal (P less than 0.01) whereas the GH-secreting pituitary tumours showed no effect when tested with from 2 to 25 ngEq/ml (n = 8, 129, NS). The effect of IGFs on human foetal somatotrope activity was dose-related for both the semipurified IGFs (2-25 ngEq/ml, n = 16, 33) and IGF-I or IGF-II (10-100 ng/ml; n = 3, 18).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of dehydroepiandrosterone (DHA) and LRH on estrogen and progesterone production by cultured placental cells has been investigated. Placental monolayer cultures were established by trypsin dispersion of term placental villi and were maintained for 5 days in culture medium containing either no steroid or 10(-7) - 10(-5)M DHA, DHA sulfate (DHAS), or 16 alpha-hydroxy-DHA (16 alpha-OH DHA), in all cases with and without the addition of 2 X 10(-7) or 2 X 10(-6)M LRH. The cultures were changed every 24 h, and the media collected were analyzed for estrogen and progesterone by RIA. 17 beta-Estradiol production was dependent on the presence of DHA or DHAS in the medium and increased in proportion to the concentration of precursor added. Similarly, estriol was produced in proportion to the amount of 16 alpha-OH DHA added to the medium. At the same time, high concentrations (10(-5) M) of DHA and DHAS, but not 16 alpha-OH DHA, markedly suppressed progesterone production. LRH had an inhibitory effect on both progesterone and estrogen output by the cultures. These studies suggest that not only the fetus, through its increasing adrenal production of DHA and DHAS toward term, but the placenta itself, through its production of LRH, could modulate placental steroid synthesis.

Hormone production in the human feto-placental unit has been studied extensively yet relatively little is known about the regulatory mechanisms involved. A tissue culture approach has been used to examine the effect of potential controlling factors on steroid production by the human mid-term fetal adrenal and mid-term and term placenta. Adrenal. The pituitary peptides corticotropin (ACTH) and alpha-melanocyte-stimulating hormone (alpha-MSH) had the most significant influence on adrenal steroidogenesis in both the fetal and definitive zones. Their effects were not identical: they enhanced dehydroepiandrosterone sulphate (DHA-S) production in a comparable manner but alpha-MSH had much less of a stimulatory effect on cortisol biosynthesis. Medium from homologous fetal pituitary cultures mimicked the effects of alpha-MSH rather than ACTH. Homologous placental culture medium and progesterone enhanced only cortisol production and only in the fetal zone cells. These results demonstrate that specific fetal pituitary and placental factors influence fetal adrenal activity and suggest a functional zonation of the fetal adrenal. Placenta. DHA, DHA-S and 16-hydroxy-DHA stimulated oestrogen biosynthesis while high concentrations of DHA and DHA-S (but not 16-hydroxy-DHA) inhibited progesterone production. Luteinizing hormone-releasing hormone (LRH) inhibited both oestrogen and progesterone biosynthesis. Placental steroidogenesis can therefore be influenced not only by the fetus, through its increasing adrenal output of oestrogen precursors, but also by factors originating within the placenta itself.

We examined the effects of transforming-growth factor-B (TGF-B) on growth ([3H]-thymidine uptake) and function (dehydroepiandrosterone sulfate [DHAS] and cortisol production) of human fetal zone adrenal cells. Results indicate that TGF-B significantly inhibits, in a dose-related manner, both basal and epidermal growth factor (EGF)-stimulated cell growth: IC50 = 0.1-0.25 ng/ml. EGF is ineffective in overcoming the inhibitory effect of TGF-B, suggesting a noncompetitive antagonism between the two factors. Also, the inhibitory effect of TGF-B is additive to that of adrenocorticotropic hormone (ACTH). On the other hand, TGF-B (1 ng/ml) does not significantly change basal or ACTH-stimulated DHAS or cortisol secretion. We conclude that, unlike its effect on other steroid-producing cells, TGF-B inhibits growth of fetal zone cells and does not appear to have a significant inhibitory effect on steroidogenesis.