Sex-specific differences in rabbit fetal lung maturation in response to epidermal growth factor.

Males and females exhibit different stages of lung development at the same gestation with males lagging behind. We hypothesized that one of the mechanisms responsible for the sex-specific difference in fetal lung maturation is a delay in the onset of epidermal growth factor (EGF) activity in the male fetal lung. EGF influences growth and differentiation during development. We studied the effects of EGF on the incorporation of glycerol into lamellar body disaturated phosphatidylcholine (DSPC) in sex-specific fetal rabbit lung explants prepared at 21 and 24 days gestation (term 31 days). The explants were maintained in Waymouth's media + 10% stripped fetal calf serum with or without EGF (10 ng/ml). The incorporation of [1,3-14C]glycerol into lamellar body DSPC was assessed after 3, 5, or 7 days of culture. Female lung explants prepared at 21 days of gestation had increased incorporation of glycerol into DSPC over time in response to EGF treatment. Male lung explants prepared at 21 days did not respond to EGF treatment. In explants prepared at 24 days gestation, baseline glycerol incorporation into DSPC was higher in female as compared to male fetal lung explants. EGF-responsiveness was also sex-specific in these more mature explants, with the male explants now responding to EGF with a consistent increase in the incorporation of glycerol into lamellar body DSPC. We conclude that one of the mechanisms responsible for the lag in male fetal lung development is a delay in the onset of EGF activity.     

Regulation of the synthesis of the major surfactant apoprotein in fetal rabbit lung tissue

Antibodies directed against the major apoprotein of rabbit lung surfactant, a 29-36-kDa glycoprotein, were used to study changes in the levels of translatable surfactant apoprotein mRNA in rabbit lung tissue during development, as well as the effects of cortisol and cyclic AMP analogues on the levels of surfactant apoprotein and its mRNA in fetal rabbit lung tissue in organ culture. The major surfactant apoprotein and its mRNA were undetectable in lung tissues of 21-day gestational age fetal rabbits. Translatable mRNA specific for the major surfactant apoprotein was first detectable in lung tissues of 26-day fetuses, increased 25-fold on day 28, reached peak levels at day 31, and declined after birth. Incubation of 21-day fetal rabbit lung explants with cortisol in serum-free medium resulted in an increase in the specific content of the 29-36-kDa apoprotein. Cyclic AMP analogues and forskolin, an activator of adenylate cyclase, also caused a marked increase in the accumulation of surfactant apoprotein. When fetal lung explants were incubated with cortisol and dibutyryl cyclic AMP in combination, the specific content of the surfactant apoprotein was increased to levels greater than that of explants treated with either cortisol or dibutyryl cyclic AMP alone. These effects of dibutyryl cyclic AMP and cortisol on surfactant apoprotein accumulation were associated with comparable changes in the levels of translatable surfactant apoprotein mRNA. Thus, we have shown for the first time that the induction of pulmonary surfactant apoprotein synthesis during differentiation in vitro and in vivo is associated with an increase in the level of translatable mRNA and that cortisol and cyclic AMP increase both the accumulation of the major surfactant apoprotein and the corresponding mRNA in fetal rabbit lung tissue in vitro.     

Hormonal regulation of beta-adrenergic receptors in fetal rabbit lung in organ culture

Explants of fetal rabbit lung were established on the 25th day of gestation. These were maintained in serum-free medium for periods up to 10 days. During this time, the cultures exhibited morphological changes typical of terminal lung differentiation. Morphological evidence was also obtained for synthesis and secretion of pulmonary surfactant in these explants. beta-Adrenergic receptors were identified in these lung explants. Exposure of the explants to 10(-7)M dexamethasone on the third day of culture resulted in a significant increase in the number of beta-adrenergic receptors in the tissue without a change in receptor affinity. The effect of dexamethasone in organ culture was dose-dependent, a maximum increase in receptor number being observed within 48 hours of incubation with a hormone concentration of 1 x 10(-7)M. Exposure of the explant tissue to 1 x 10(-7)M triiodothyronine resulted in no significant increase in the concentration of beta-adrenergic receptors and no change in receptor affinity. These results suggest that glucocorticoids may potentiate the effects of beta-adrenergic agents in the fetal lung by increasing the numbers of their receptors. The effects of triiodothyronine upon the fetal lung do not appear to be mediated by this mechanism.     

Metabolism and function of platelet-activating factor in fetal rabbit lung development

Previously, platelet-activating factor (PAF, PAF-acether, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) had been identified in association with a lamellar-body-enriched fraction of human amniotic fluid obtained from women in labor. In consideration of the fact that fetal lung is the source of lamellar bodies, we have investigated the capacity of the developing lung to synthesize PAF. The specific activity of the PAF biosynthetic enzyme, 1-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase, increased from 116 pmol/min per mg protein in day 21 fetal rabbit lung to 332 pmol/min per mg protein by day 31. Although this enzymatic activity in fetal kidney also increased, it never reached the level found in lung. In contrast, the actyltransferase activity decreased by 80% in fetal liver between days 21 and 31. The acetyltransferase activity in lung was primarily localized in the microsomal fraction (105 000 X g pellet); however a significant proportion of the activity was found in the 18 000 X g pellet. The specific activity of acetyltransferase in adult alveolar type II rat pneumonocytes was significantly higher than that of adult rat lung or rat alveolar macrophages, suggesting that type II cells make a significant contribution to the actyltransferase activity of lung tissue. PAF acetylhydrolase remained relatively constant throughout the gestation in all tissues. The concentration of PAF in the fetal lung increased by 3-fold from 12 to 35 fmol/mg protein, between day 21 and day 31 of development. The concentrations of the PAF precursors, 2-lyso-PAF (1-alkyl-2-lyso-sn-glycero-3-phosphocholine) and the 2-acyl derivative, were several orders of magnitude higher than the PAF concentration. The pulmonary glycogen content decreased from 163 at day 21 to 35 micrograms/mg protein at day 31 of gestation. We suggest that the increase in PAF concentration may participate in the regulation of glycogen breakdown in fetal lung as it does in perfused rat liver (Shukla, S.D., Buxton, D.B., Olson, M.S. and Hanahan, D.J. (1983) J. Biol. Chem. 258, 10212-10214). The formation of PAF in the developing lung and its secretion, in association with lamellar bodies, into amniotic fluid is discussed in relation to parturition.     

Neuro-epithelial bodies in organ cultures of fetal rabbit lungs

Lung explants from fetal rabbit at the late glandular stage of development (20 days' gestation) and near term (31 days' gestation) were maintained in organ culture for up to 22 days. They were studied by light and electron microscopy to determine whether neuro-epithelial bodies (NEB) of the lung retain structural integrity in vitro. Cultured NEB retained argyrophilia and specific amine fluorescence after formaldehyde condensation. Their ultrastructural morphology showed some differences from that of uncultured NEB: the terminal axons had degenerated and the secretory granules (dense-core vesicles, DCV) were slightly larger, more pleomorphic, more electron-dense, and redistributed throughout the cytoplasm rather than being confined chiefly to the basal regions. These changes, together with hypertrophy of Golgi zones, suggest increased synthesis and storage of secretory products in the DCV during culture. In NEB from near-term explants cultured for 7 days and incubated with reserpine, the core of DCV decreased in size and electron-density and became finely granular, a sign of amine release. Ca++ ionophore No. A-23187, also, induced changes in the ultrastructure of DCV, suggesting that the secretory process in lung neuro-endocrine cells, as in other secretory cells, is Ca++-dependent.     

The effect of human urogastrone on lung phospholipids in fetal rabbits

Previous in vivo studies have demonstrated that mouse epidermal growth factor (EGF) can enhance fetal lung maturation. We have examined the effect of urogastrone, the human equivalent of mouse EGF and a related growth factor, on the phospholipid profile of fetal rabbit lung lavage and its action on fetal rabbit Type II pneumocytes in culture. Urogastrone (1 or 8 micrograms) given i.p. to fetal rabbits on day 25 of gestation resulted in increased total phospholipid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine contents, increased phosphatidylinositol and phosphatidylethanolamine as a proportion of phospholipid and decreased sphingomyelin as a proportion of phospholipid in lung lavages on day 28. These changes were unaccompanied by alterations in body weight or lung weight, DNA or protein concentrations. Urogastrone (16 micrograms) resulted in increased fetal deaths. Phospholipid profiles on day 27 were unchanged after fetal administration of urogastrone (1 microgram) on day 25. Urogastrone (0.01 and 0.1 ng/ml) added to fetal rabbit Type II pneumocytes in culture for 24 h enhanced the incorporation of radiolabelled choline and thymidine into phosphatidylcholine and DNA respectively. These findings indicate that human urogastrone can alter the phospholipid composition of the rabbit lung in a similar manner to that which occurs during maturation of the lung surfactant system in late pregnancy. This effect can be achieved, at least in part, by a direct action on Type II pneumocytes.     

Ontogeny of pyruvate dehydrogenase complex and key enzymes involved in glycolysis and tricarboxylic acid cycle in rabbit fetal lung, heart, and liver

The metabolic pathways by which the glycogen is utilized by fetal tissues is not well established. In the present study the ontogeny of seven key enzymes involved in glycolysis and the tricarboxylic acid cycle has been established for rabbit fetal lung, heart, and liver. In the fetal lung the activities of phosphofructokinase, pyruvate kinase, lactic dehydrogenase, citrate synthase, and malate dehydrogenase increase from day 21 to 25. Thereafter the levels either drop to day 19 levels or do not change. The isocitrate dehydrogenase activity continues to increase from day 19 of gestation to maximum level on day 31 of gestation. In fetal heart the pattern of activity is similar, but in fetal liver most of the enzymes reach maximum levels earlier and, with the exception of pyruvate kinase, do not show a significant fall in activity near term. The pattern of development of pyruvate dehydrogenase complex is different; maximum activity is observed on day 27 in fetal lung and heart and on day 21 in fetal liver. These results indicate that all three fetal tissues can oxidize glucose. Also, the accumulation of glycogen, particularly in fetal lung, appears to ensure that at specific times during gestation adequate quantities of energy (ATP) and substrates, required for surfactant phospholipid synthesis, are available independent of maternal supply of glucose or during brief episodes of hypoxia.     

A simplified method for culture of human fetal heart tissue

Human fetal heart tissue obtained consequent to suction termination of pregnancy between 6 and 12 weeks of gestation were cultured as explants and maintained in a viable state, with spontaneous contractions up to 75 days. Ultrastructural morphology of the explant revealed that the cells remained healthy up to 21 days in culture. The model can therefore be used for experimental studies during the first 3 weeks in culture.     

Protein profiling the effects of in vitro hyperoxic exposure on fetal rabbit lung

The aim of this study was to test the hypothesis that acute in vitro exposure of prematurely delivered fetal rabbit lungs to hyperoxic conditions will induce the expression of an adaptive cassette of proteins that mediates antioxidant and inflammatory processes. To test this hypothesis, ex situ fetal rabbit lung explants were prepared from New Zealand white rabbits delivered by cesarean section on day 29 of gestation and incubated under air (21% O2; 5% CO2) or hyperoxic (95% O2; 5% CO2) atmospheres. Total tissue protein was extracted following incubation and subjected to 2-DE. Using this technique, 1500-2000 protein spots were resolved per gel. Treatment-dependent, differentially expressed proteins were identified by image analysis (Melanie II) and MALDI-TOF MS and MALDI-MS/MS. The analysis identified 12 protein spots that were differentially expressed by 1.5-fold or more (p<0.05) by exposure to hyperoxic conditions. Six of these differentially expressed proteins were identified as vimentin, annexin I, inorganic pyrophosphatase, prohibitin, an N-terminal fragment of ATP synthase and heat shock protein 27. The data obtained are consistent with the roles of these proteins in mediating cellular response to oxidative stress and in regulating cell proliferation.     

Effect of cortisol on the synthesis of lamellar body glycerophospholipids in fetal rabbit lung tissue in vitro

The effect of cortisol on the rate of choline incorporation into tissue phosphatidylcholine was investigated in lung explants from fetal rabbits of 19-28 days gestational age. The explants were incubated in medium with or without fetal calf serum for up to 7 days. When lung tissues were incubated in serum-free medium, a stimulatory effect of cortisol on tissue phosphatidylcholine synthesis was found in explants from 21-, 24-, 26- and 28-day fetal rabbits; a stimulatory effect of cortisol was observed in 19-day fetal lung explants only if fetal calf serum was present in the culture medium. To assess directly the effect of cortisol on the synthesis of lamellar body phosphatidylcholine, choline incorporation into phosphatidylcholine associated with a purified lamellar body fraction isolated from lung explants of 21- and 28-day fetal rabbits was also investigated. Cortisol caused a marked stimulation of synthesis and accumulation of lamellar body phosphatidylcholine in lung explants from both 21- and 28-day fetal rabbits. The magnitude of the stimulatory effect of cortisol on the rate of synthesis of lamellar body phosphatidylcholine was always greater than the effect of cortisol on the rate of choline incorporation into lipids of tissue homogenates. The relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were also significantly altered in lung explants from 21- and 28-day fetal rabbits by cortisol treatment. Lamellar bodies that were formed initially in the fetal lung explants were enriched in phosphatidylcholine and phosphatidylinositol and had a relatively low phosphatidylglycerol content. With cortisol treatment there was a decrease in the relative rate of synthesis of lamellar body phosphatidylinositol and an increase in the relative rate of synthesis of phosphatidylglycerol. The stimulatory effect of cortisol on the synthesis of lamellar body phosphatidylcholine was observed at an earlier time-point of incubation than was the effect of cortisol on the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol. The temporal sequence of the cortisol-induced changes in the synthesis of lamellar body glycerophospholipids, therefore, reflects that which occurs with maturation in vivo.     

Fetal lung disaturated phosphatidylcholine. Ostensible increase following exposure to dexamethasone

Fetal rat lung removed at 15 days gestation and placed in organ culture incorporates choline into phosphatidylcholine. Addition of 10(-9) M dexamethasone resulted in increased rates of choline incorporation per micrograms protein after both 6 and 12 days culture. This concentration of dexamethasone did not increase tissue phosphatidylcholine or disaturated phosphatidylcholine. Thus, at a culture time when dexamethasone had a significant effect on choline incorporation, there was no change in either the total phospholipid or disaturated phosphatidylcholine content of the lung tissue. The transplacental administration of dexamethasone decreased fetal lung DNA and phospholipid content. At the mid-range dosage tested (400 micrograms), dexamethasone depressed DNA (51%) appreciably more than total phosphatidylcholine (28%) and disaturated phosphatidylcholine (33%). These results show that the hormone does not increase the total amount of surfactant per lung. The increased disaturated phosphatidylcholine per mg DNA results in an ostensible beneficial effect of dexamethasone on surfactant and may reflect an increased proportion of Type II cells in fetal lung both in vitro and in vivo following hormone exposure. Disaturated phosphatidylcholine per Type II alveolar cell is no doubt increased but the trade-off is fewer total cells in the lung.     

Ultrastructural study of Pneumocystis carinii in explant cultures of rabbit lung and in cultures with and without feeder cells

Pneumocystis carinii-parasitized lung explants were obtained from corticoid-treated rabbits and maintained in vitro. Twenty-one days after the beginning of explant cultures, the ultrastructural morphology of trophozoite, precyst and cyst forms was normal as compared to the in vivo ultrastructure of P. carinii from infected rabbit. However, after the 36th day, only altered forms of P. carinii were observed. Lung tissue showed only minor alterations. Intracytoplasmic lamellar inclusions were observed in type 2 alveolar cells from which they were released. While the total number of parasites increased approximately 4-fold from day 0 to day 41, trophozoite counts increased approximately 6 times. Pneumocystis cells from inocula and supernates of cultures with and without Vero cells showed important ultrastructural alterations.     

Measurement by radioimmunoassay of casein content in rabbit mammary gland during pregnancy and after prolactin stimulation in organ culture

A specific homologous radioimmunoassay was developed to measure rabbit beta-casein in rabbit mammary gland with a sensitivity of 0.5 ng/ml protein. It was used to measure casein concentration during pregnancy and in organ culture of mammary gland explants. Casein was detectable in virgin mammary glands, showed a small increase during the first half of pregnancy, increased more than 20-fold between Days 21 and 27, and diminished somewhat on the first days of lactation. After 24 hr of culture, mammary gland explants had no detectable casein, but the addition of increasing concentrations of prolactin to a culture medium which contained insulin (5 micrograms/ml) and cortisol (0.5 microgram/ml) induced a regular increase in the casein content of the tissue. Casein started to increase when 10 ng/ml of prolactin was present and maximal values were achieved for 100 ng/ml of the hormone.     

Evidence for lactate utilization for fetal lung glycogen synthesis

Fetal rabbit lungs from 23 day gestation animals were used to investigate the potential role of lactate as a substrate for fetal lung glycogen synthesis. Fetal lactate dehydrogenase activity was approximately twice that found in the adult lung, while the activity of phosphoenolpyruvate carboxykinase was elevated fourfold over the adult value. Pyruvate carboxylase activities were similar in both fetal and adult lungs. Studies employing fetal lung explants in organ culture indicated that the presence of both glucose and lactate may be necessary for glycogen accumulation in the developing fetal lung. These data support the hypothesis that lactate is an important precursor for fetal lung glycogen.     

A role for cytidine monophosphate in the regulation of the glycerophospholipid composition of surfactant in developing lung

The concentration of cytidine monophosphate (CMP) in lung tissue was found to increase during rabbit lung development from 12 nmol/g of tissue on day 23 of gestation to 52 nmol/g in the adult. The concentrations of cytidine triphosphate in lung tissue decreased from 113 nmol/g of tissue on day 23 to 63 nmol/g of tissue in the adult. The concentration of CDP-choline increased from 14.9 nmol/g at day 21 of gestation to 38.4 at day 26 and decreased subsequently in the newborn (6.5 nmol/g) and adult (9.7 nmol/g). The increase in the concentration of CMP in lung appeared to be organ and nucleotide specific since there was no increase in the concentration of CMP in liver tissue, and the concentration of AMP in the lung tissue did not increase with development. A function for CMP in regulating the availability of CDP-diacylglycerol is proposed to account for the changes in the glycerophospholipid composition of lung surfactant which occur during development.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

The effect of cortisol on rabbit fetal lung maturation in vitro

Explants of lung tissue from 19-day gestational age fetal rabbits were maintained in organ culture in medium with or without fetal calf serum for 1 to 11 days. Based on the results of biochemical and morphological studies it was apparent that the type II pneumonocyte differentiated in vitro at a time similar to that which occurs with maturation in vivo. The epithelial cells of the presumptive alveoli were undifferentiated at the start of incubation, but within 9 days developed increased amounts of Golgi apparatus and rough endoplasmic reticulum, many microvilli on the luminal surface and numerous lamellar bodies. Secreted lamellar bodies and tubular myelin figures were observed in the lumina of cultured explants. The incorporation of [³H]choline into phosphatidylcholine by lung tissue explants maintained in medium containing 10% fetal calf serum remained relatively constant for 7 days of incubation but thereafter increased two-fold. When explants were maintained in fetal calf serum-containing medium and cortisol (10^?7M) or betamethasone (10^?7M), the incorporation of choline into phosphatidylcholine was two to three times greater than that of explants maintained in serum-containing medium without cortisol. When explants of fetal lung tissue were incubated in the presence of cortisol without fetal calf serum there was no stimulatory effect of cortisol on phosphatidylcholine biosynthesis. Therefore, serum cofactors are necessary for the stimulatory effects of cortisol on fetal lung development. The specific activity of phosphatidate phosphohydrolase (PAPase) increased to very high levels during the culture period. In the presence of serum, cortisol or betamethasone had no effect on the specific activity of phosphatidate phosphohydrolase.     

Identification and ontogenesis of beta-adrenergic receptors in fetal and neonatal rabbit myocardium

In adult animals, beta-adrenergic receptors are involved in the regulation of myocardial contractility and heart rate. Their properties in the fetal and early neonatal period have not been adequately investigated. We have directly characterized beta-adrenergic receptors in rabbit fetal and neonatal myocardial membranes by a radioligand binding assay utilizing 125I-hydroxybenzylpindolol (I-HYP), a potent, non-specific, beta-adrenergic antagonist, I-HYP binding sites were detected as early as the 21st day of gestation (term 31 days). The binding was rapid, saturable and reversible. The dissociation constant, KD, as determined by Scatchard plots, ranged from 30 pM to 50 pM. There was a progressive increase in the density of the receptor sites with advancing gestation. Competition studies with beta-agonists and antagonists showed that the order of potency in inhibiting I-HYP binding was consistent with a beta 1-subtype of beta-adrenergic receptors. We conclude that the progressive increase in density of beta-adrenergic receptors in rabbit fetal myocardium parallels similar maturational processes occurring in other tissues with advancing gestation. It may also account for the reported developmental changes in fetal myocardial contractility.