Charlotte L. Branchaud

Short term tissue culture of human midterm and term placenta: Parameters of hormonogenesis

Abstract Monolayer cultures of human midterm and term placentae have been established following trypsin dispersion of placental minces. Maintenance of endocrine function was monitored by the concentrations of specific hormones in the culture media. At either gestational age the cultures 1) secrete estradiol-17β(1) and estrone (in a ratio of about 1: 20) and aromatize 3H- or 14C-dehydroepiandrosterone sulfate and 14C-androstenedione, estrogen production being markedly enhanced by addition of dehydroepiandrosterone (10−6M) to the culture medium; 2) metabolize 3H-pregnenolone to progesterone and 14C-cortisol to cortisone; and 3) produce increasing amounts of chorionic gonadotropin and decreasing amounts of placental lactogen during the first week in culture. It is proposed that the model is highly suited to the study of factors affecting hormonogenesis by the human placenta whether they be of maternal or of fetal origin

Fetal Metabolism of Cortisol

Publisher Summary This chapter discusses the sources, fate, and significance of fetal cortisol with particular emphasis on human species. During most of gestation, diffusible cortisol levels remain low, favoring rapid growth. The rapid growth is aided by rapid inactivation to cortisone in all fetal tissues, particularly the placenta, which prevents the bulk of maternal cortisol crossing the placenta from reaching the fetus in an active form. Thus, shielded from the high cortisol levels in the mother where the main thrust of metabolism is directed toward providing the fetus with nutrients, the fetus is able to maintain autonomy with respect to its cortisol production. At midterm, much of fetal cortisol synthesis probably occurs by the adrenal hydroxylation of progesterone. The striking growth peak of the fetal adrenal in relation to total body weight at 15 weeks may be due to human chronic gonadotropin (hCG) as it appears to occur shortly after the peak of fetal hCG seen at this time, but hCG has little effect on steroidogenesis.

Characterization of the 21-yl sulfates of llβ,17α,21-trihydroxypregn-4-ene-3, 20-dione; 17α,21-dihydroxypregn-4-ene-3,11,20-trione; llβ,21-dihydroxypregn-4-ene-3, 20-dione; 21-hydroxypregn-4-ene-3,11,20-trione and 21-hydroxypregn-4-ene-3,20-dione in human cord plasma

Abstract The 21-yl sulfates of 113,17α,21-trihydroxypregn-4-ene-3,20-dione (cortisol); 17α,21-dihydroxypregn-4-ene-3,11,20-trione (cortisone); llβ, 21-dihydroxypregn-4-ene-3,20-dione (corticosterone); 21-hydroxypregn-4-ene-3, 11,20-trione (11-dehydrocorticosterone) and 21-hydroxypregn-4-ene-3,20-dione (11-deoxycorticosterone) were identified in pools of human mixed cord plasma by a method combining double isotope assay with crystallization. The data imply the presence of these conjugates in the fetal circulation, at term.

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.

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

Summary: 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 radioim-munoassay 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.

A serum-free system for culturing human placental trophoblasts

SummaryWe have compared hormone production by early gestation and term human placental trophoblasts cultured in Hams 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 Hams F10, but production was maintained at a stable level for at least 7 d longer than the cells in Hams. Estradiol production from 10−6M 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 Hams 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 Hams 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 differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.

Exploration of the human fetal pituitary adrenal axis: stimulation of cortisol and dehydroepiandrosterone sulfate biosynthesis by homologous pituitary in organ culture.

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.

Doxapram metabolism in human fetal hepatic organ culture

The biotransformation of doxapram, a respiratory stimulant was studied with use of explants from human fetal livers (n = 15 fetuses) obtained from therapeutic abortions (gestational age, 10 to 20 weeks). Explants were cultured in Leibowitz medium and the media from cultured samples were collected before and at 3, 6, 12, and 24 hours after incubation with 2.5, 5.0, and 10 μg/ml doxapram. The concentrations of doxapram and its metabolites (AHR 0914, an analog of doxapram, AHR 5955 or ketodoxapram, and AHR 5904) were measured by high pressure liquid chromatography. Explant histopathology and alkaline phosphatase activity showed no direct toxic effects of the drug on liver tissue. The fastest rate of doxapram metabolism occurred during the first 3 hours of incubation (198 ± 73.3, 438 ± 63.3, and 538 ± 62 ng/mg/hr liver protein at doxapram concentrations of 2.5, 5.0, and 10.0 μg/ml, respectively). At 3 hours of incubation, the amount of doxapram metabolized (nanogram per milligram of liver protein) was significantly higher (p < 0.01) at doxapram concentrations of 10.0 (1616 ± 186) and 5.0 μg/ml (1315 ± 190) than at 2.5 μg/ml (594 ± 220). The oxidative pathway producing keto‐doxapram, or AHR 5955 and AHR 5904, is more active than the de‐ethylation producing the analog of doxapram AHR 0914. Data indicate substantial metabolism of doxapram by the human fetal liver.

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 Hams 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 Hams 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.