Effects of growth hormone (GH)-releasing factor and somatostatin on GH secretion from early to midgestation human fetal pituitaries.

Using explant cultures of human anterior pituitary glands (9-19 weeks fetal age) and an acute (3-h) test protocol, we investigated fetal somatotrope responsiveness to human GH-releasing factor [hGRF-(1-44)] and somatostatin [SRIF-(1-14) and SRIF-(1-28)] as a function of age. Ontogenic data were analyzed using three age groups: 9-10, 12-13, and 15-19 weeks fetal age. Both daily (24-h) and acute test (3-h) basal GH secretion increases as a function of fetal age, with the greatest increase occurring after 12-13 weeks; however, the 3 h/24 h secretion ratio remains unchanged, at approximately 12%. GRF (0.1-10 nM) stimulates GH release in a dose-related fashion, regardless of fetal age; there is a significant increase in the response to both 1 nM (P < 0.05) and 10 nM (P < 0.01) GRF between 9-10 and 15-19 weeks fetal age and to 10 nM GRF (P < 0.05) between 12-13 and 15-19 weeks. Pretreatment of cultures (9-19 weeks) with 1 or 10 nM GRF for 24 h does not alter basal 3-h GH secretion, but significantly decreases subsequent responses to 1 or 10 nM GRF, respectively (P < 0.01). Pretreatment with 1 nM GRF does not alter a subsequent 3-h response to 10 nM GRF. SRIF-(1-14) (1-100 nM) causes a dose-related inhibition of basal GH secretion from as early as the ninth week of fetal life; there is a small age-related increase in the somatotrope response to 100 nM SRIF-(1-14) between 12-13 and 15-19 weeks fetal age (P < 0.05). In a group of 11- to 14-week-old fetal pituitaries, SRIF-(1-28) had a significantly (P < 0.05) greater inhibitory effect than SRIF-(1-14) at both 1 and 10 nM; the two peptides decreased basal GH secretion to a similar extent at 100 nM (52.8 +/- 4.0% of control value; P < 0.01). SRIF-(1-14) (10 and 100 nM) does not significantly alter 10 nM GRF-stimulated GH release from 9- to 10-week-old fetal pituitaries. However, by 12-13 weeks, 10 nM SRIF-(1-14) reduces GRF-stimulated GH secretion by 60% (P < 0.01), while 100 nM SRIF-(1-14) decreases it by 80% (P < 0.01); similar inhibitory effects are observed with 15- to 19-week-old fetal somatotropes.(ABSTRACT TRUNCATED AT 400 WORDS)

In vitro modulation of growth hormone (GH) secretion from early to midgestation human fetal pituitaries by GH-releasing factor and somatostatin: role of Gs-adenylate cyclase-Gi complex and Ca2+ channels.

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)

Effect of maternal serum on viability and function of early human placental explants.

Fetal bovine serum (FBS) is frequently used to supplement chemically defined media such as Ham's F10 when studying placental explant cultures. However in vitro production of hormones is usually declining by the 2nd or 3rd day and is short-lived (7 to 10 days). In this study we explored the use of human maternal serum (HMS) from early gestation as the medium supplement to Ham's F10. Early placental hormone production was compared using two concentrations of FBS and HMS. On Day 3 of incubation, progesterone production in 10% HMS was 12-fold increased over that in 10% FBS, estradiol production was increased 10-fold, and ßhCG production more than 3-fold. When the serum concentrations were increased to 40%, the results in all cases were similar to those at 10%. Preliminary characterization studies revealed that the stimulatory activity of HMS is heat-labile, neither extractable into organic solvent (diethyl ether) nor dialyzable, suggesting that it is protein in nature. In a long-term incubation, compared with FBS (7 days), HMS permitted survival of culture up to 30 days, judged both histologically and biochemically. We conclude that HMS provides substance(s), probably protein in nature, not present in FBS or non-pregnant human serum, which are important for human placental viability and function in vivo.

A serum-free system for culturing human placental trophoblasts.

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.

Effect of placental factors on growth and function of the human fetal adrenal in vitro.

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.

Growth-inhibitory effect of TGF-B on human fetal adrenal cells in primary monolayer culture.

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.

The effects of human chorionic gonadotropin on growth and steroidogenesis of the human fetal adrenal gland in vitro.

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.

Regulation of growth hormone secretion from human fetal pituitaries: interactions between growth hormone releasing factor and somatostatin.

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.

Contribution of exogenous progesterone to human adrenal cortisol synthesis in vitro: a comparison of early gestation fetal and adult tissues.

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.

Insulin-induced receptor regulation in early gestation and term human placental cell cultures.

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.

Functional zonation of the midgestation human fetal adrenal cortex: fetal versus definitive zone use of progesterone for cortisol synthesis.

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.

Effect of caffeine on thyroid and pituitary function in newborn rats.

The possibility that caffeine, a central nervous system stimulant used in neonatal apnea, may produce acute or chronic changes in growth hormone (GH), thyroxine (T4) and thyrotropin (TSH) was studied in the newborn rat. Five-day-old rats were separated into three groups: control (0) group receiving saline, Group I (low dose caffeine) receiving 5 mg/kg and Group II (high dose caffeine) receiving 50 mg/kg. Acute effects were studied at 2, 4, and 24 h after injection. Chronic effects were studied 24 h after the last of 10 daily injections. GH, T4, and TSH were measured by radioimmunoassay and caffeine by high pressure liquid chromatograph. GH was increased at all times and all doses after a single injection of caffeine. After chronic therapy, the increase in GH was small, suggesting depletion of pituitary reserve. A high dose of caffeine had a biphasic effect on T4 with an increase at 4 h and a decrease at 24 h. Thyrotropin-releasing hormone (TRH)-induced TSH release at 24 h was not influenced by caffeine administration. Chronic caffeine therapy stimulated both T4 and TSH; however, TRH-stimulated TSH release was decreased, suggesting that chronic therapy may blunt pituitary TSH response.

Progesterone and estrogen production by placental monolayer cultures: effect of dehydroepiandrosterone and luteinizing hormone-releasing hormone.

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.

Cortisol-cortisone interconversion in human fetal lung: contrasting results using explant and monolayer cultures suggest that 11 beta-hydroxysteroid dehydrogenase (EC 1.1.1.146) comprises two enzymes.

Since cortisol (F) can influence fetal lung maturation, alterations in its intracellular metabolism may be important. In fetal lung at midgestation, both the in vivo studies of Pasqualini et al. and the in vitro studies of Murphy showed the activity of the enzyme 11 beta-hydroxysteroid dehydrogenase (EC 1.1.1.146) to be strongly oxidative, converting F to its inactive analog cortisone (E). By contrast, Smith et al. observed only reductive activity (E to F) in monolayer cultures. The object of this study was to resolve this discrepancy. Human fetal lung (HFL) of gestational age 9-13 weeks was grown in monolayer cultures or as explants on grids in Ham's F-10 medium plus 10% fetal calf serum and [3H]F and [14C]E (12-20 ng/ml of each). Extracts of media were chromatographed to separate the steroids, and the percent conversion was calculated. Explants converted F to E 20-40% and maintained day 1 levels for at least 7 days of culture. E to F conversion was initially low (1-5%) and rose to 14-20% by the end of culture, corresponding to fibroblast-like outgrowth. In monolayer cultures, which appeared to consist almost entirely of fibroblast-like cells, F to E conversion was less than 10% by 5 days, while E to F conversion steadily increased to 20-85% and plateaued at confluence. Homogenates of fresh tissue demonstrated F to E but no E to F conversion, even in the presence of cofactors. Thus, it appears that when tissue structure is maintained as in the explants, HFL cells oxidize F to E by a unidirectional enzyme; activation of E to F is only expressed by HFL fibroblast-like cells in culture and does not reflect the in vivo situation.

Regulation of hormone production in the human feto-placental unit.

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.