The influence of hormones on the biochemical development of fetal rat lung in organ culture

375

Biochimica et Biophysics Acta, 575 (1979) 375-383 0 Elsevier/North-Holland Biomedical Press

BBA 57478

THE INFLUENCE OF HORMONES ON THE BIOCHEMICAL DEVELOPMENT OF FETAL RAT LUNG IN ORGAN CULTURE I. ESTROGEN

IAN GROSS *, CHRISTINE and SEAMUS A. ROONEY

M. WILSON,

LINDA

D. INGLESON,

ARLETTE

BREHIER

Division of Perinatal Medicine, Departments of Pediatrics and Obstetrics and Gynecology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 (U.S.A.) (Received

March

14th,

1979)

Key words: Estrogen; Phospholipid;

Glycogen;

(Fetal rat lung)

Summary It was recently demonstrated that 17&estradiol enhances phosphatidylcholine synthesis in fetal lung in vivo. In order to determine whether estrogen acts directly on the lung, we examined the influence of 17/3-estradiol on phospholipid and glycogen metabolism in explants of 18 day fetal rat lung in organ culture. Exposure of the explants to 17/3-estradiol resulted in significant stimulation of [Me-3H] choline incorporation into phosphatidylcholine, disaturated phosphatidylcholine and sphingomyelin. Incorporation of [3H]acetate into total phospholipid was increased by 63% (P < 0.001). Incorporation into individual phospholipids was similarly increased, except for that into phosphatidylglycerol which was enhanced by 111% (P < 0.005). When the data were expressed as the percentage of radioactivity from [3H]acetate in the various phospholipid fractions, it was found that there was a 29% increase in the phosphatidylglycerol fraction (P < 0.005) and a 12% reduction in phosphatidylinositol plus phosphatidylserine combined (P < 0.005). There was no significant change in the percentage of radioactivity in any of the other phospholipid fractions, or in the activity of any of the enzymes of phospholipid synthesis examined. Estrogen treatment also resulted in a decrease in the glycogen content (P < 0.05), but no change in the protein/DNA ratio. These data indicate that estrogen acts directly on the fetal lung and

* To whom correspondence and reprint requests should be addressed: Dr. Ian Gross, Department Pediatrics, Yale School of Medicine, 333 Cedar Street. New Haven, CT 06510 (U.S.A.).

of

376

stimulates the incorporation of choline and acetate into phospholipids. It may specifically enhance incorporation into phosphatidylglycerol, the second most abundant phospholipid in pulmonary surfactant.

Introduction Pulmonary surfactant is the phospholipid rich material which lines the alveoli of the lungs and prevents their collapse due to surface-tension at the airalveolar interface. Disaturated phosphatidylcholine and phosphatidylglycerol are the major phospholipid components of surfactant [l]. A number of hormones and other agents have been shown to stimulate fetal lung maturation and surfactant production. These include corticosteroids, thyroxine, cyclic AMP, and possibly prolactin [Z]. Recently Khosla and Rooney [3] reported that injection of 17/3-estradiol into pregnant rabbits results in a marked increase in the surfactant content of the fetal lungs. The estradiol treated fetus had 4 times more phosphatidylcholine in the lung lavage than did the control animals and the phosphatidylcholine:sphingomyelin ratio of the lavage was significantly higher in the treated group. These findings suggest that estrogen may play a role in lung maturation and surfactant production. However they do not indicate whether estrogen is acting directly on the lung or is producing its effect via other hormones. Estrogen has been shown to stimulate the synthesis and release of prolactin by the anterior pituitary gland [4] and to enhance the synthesis of corticosteroids by the adrenal [ 51. The observed effects on the lung could therefore have been mediated by corticosteroid or prolactin. In order to determine whether estrogens act directly on the fetal lung, we have examined the influence of 17P-estradiol on phospholipid and glycogen metabolism in fetal rat lung in organ culture. Materials and Methods Organ culture. The organ culture model has been described in detail previously [6], but was modified for this series of experiments. Timed, 18-day pregnant Sprague-Dawley rats (term is 22 days) were killed by decapitation and the fetuses removed under sterile conditions. The lungs were excised and chopped into 1 mm cubes using a McIlwain tissue chopper. Approx. 20 explants were then placed on the surface of a 60-mm diameter tissue culture dish to which 2 ml Waymouth’s MB 752/l medium, containing 100 units/ml penicillin and 100 pg/ml streptomycin were added. (Waymouth’s MB 752/l medium contains 1.79 mM Choline.) No serum was added to the culture medium. The explants were incubated at 37°C in a humidified atmosphere of 95% 02/5% CO2 in a Plexiglas controlled atmosphere culture chamber (Bellco, Vineland, NJ). The explants were allowed to rest on the surface of the culture dish for 2 h to facilitate adherence of the tissue to the surface of the dish. The cultures were then rocked from side-to-side at 3 cycles/min on a rocking platform (Bellco). The effect of this was to ensure that, at any one time, half the explants were submerged in the culture medium while the other half was

377

Incubation was continued for 48 h. exposed directly to the atmosphere. Control cultures were those that contained no estrogen in the culture medium, whereas experimental cultures were exposed to 10-8-10-5 M 17P-estradiol (Sigma, St. Louis, MO), dissolved in ethanol. Biochemical analysis. After 48 h, the culture medium was aspirated and replaced with fresh medium containing either 8 /Ki [Me-3H]choline chloride, 2.23 Ci/mol, or 13.3 @i [jH]acetate, 6.4 Ci/mmol and the incubation was continued for another 4 h. The explants were then washed with ice-cold 0.9% NaCl and sonicated in 2 ml 0.9% NaCl prior to lipid analysis and protein and DNA determination. Lipid was extracted from the sonicated tissue with CHC13/ CH30H by the method of Bligh and Dyer [ 71. Phospholipids were identified by thin-layer chromatography (TLC) on silica gel (LQD plates, Quantum Industries, Fairfield, NJ) after the lipid extract had been divided into 3 aliquots. The first aliquot was separated with CHC13/CH30H/CH3COOH/Hz0 (50 : 35 : 4 : 2, v/v). This system separates lysophosphatidylcholine, sphingomyelin, phosphatidylcholine and phosphatidylinositol plus phosphatidylserine [ 81. The second plate was developed in tetrahydrofuran/methylal/CH30H/2 M NH40H (50 : 40 : 10 : 5.5, v/v). This system effectively separates phosphatidylethanolamine, cardiolipin and phosphatidylglycerol [ 81. The third aliquot was used to isolate disaturated phosphatidylcholine by a modification of the method of Mason et al. [9] as follows. Phosphatidylcholine was isolated by TLC and eluted from the gel in CHC13/CH30H/HCOOH/H,0 (97 : 97 : 2 : 4, v/v). The dried lipid was reacted with 0s04, spotted on a TLC plate and analyzed in CHC13/CH30H/ 7 M NH40H (80 : 28 : 6, v/v). This solvent mixture allows for complete separation of the disaturated species from the osmium reacted unsaturated species. After visualization with IZ, the phospholipid-containing spots were scraped off the plates into scintillation vials and the radioactivity determined in 0.5 ml HZ0 and 14.5 ml Aquasol (New England Nuclear, Boston, MA). Protein content was determined by the method of Lowry et al. [lo], glycogen content by an enzymic method described previously [ll] and DNA by a fluorimetric assay, based on binding to mithramycin (Mithracin, Pfizer, NY) [12]. Standards for these determinations were bovine serum albumin, rabbit muscle glycogen and calf thymus DNA, respectively. Explants which were used for glycogen analysis were rapidly frozen in test tubes in a solid CO,/ CzH50H bath and stored at -70” C. Enzyme assays. Explants were sonicated in chilled 0.33 M sucrose, 0.01 M Tris-HCl, 0.001 M EDTA (pH 7.4). All enzyme assays were performed in the cell-free homogenate, except for cholinephosphate cytidylyltransferase which was assayed in the 105 000 X g supernatant fraction. The activities of phosphatidate phosphatase (EC 3.1.3.4) [ll], choline kinase (EC 2.7.1.32) [11,13], cholinephosphate cytidylyltransferase (EC 2.7.7.15) [ 11,131, cholinephosphotransferase (EC 2.7.8.2) [ 141 and glycerophosphate phosphatidyltransferase (EC 2.7.8.7) and phosphatidylglycerophosphatase (EC 3.1.3.27) combined [ 151 were determined as described previously. 1-Acylglycerophosphate acyltransferase (EC 2.3.1.51) was assayed by measuring the rate of incorporation of radioactivity from [ 1-14C]palmitoyl-CoA into phosphatidic acid. The reaction mixture contained 91 mM Tris-HCl (pH 7.4), 5 PM [l-‘4C]paImitoyl-CoA (40 Wmol), 20 E.IMl-palmitoylglycerophosphate and protein in a total of 0.5 ml.

The reaction was carried out in duplicate at 37°C. O.l-ml aliquots were removed at intervals and added to CHC1JCH30H (1 : 2, v/v). Following lipid extraction by the procedure of Bligh and Dyer [7], phosphatidic acid was isolated by TLC in CHC13/CH30H/CH3COOH/Hz0 (80 : 15 : 2 : 2, v/v). The radioactivity of the phosphatidic acid spot was measured in Liquifluor (New England Nuclear). Under these conditions the rate of the reaction was linear with time for 3 min and with protein concentration up to 100 p&/ml. Lysolecithin acyltransferase (EC 2.3.1.23) was similarly assayed by measuring the rate of incorporation of [ 1-14C]palmitoyl-CoA into phosphatidylcholine. The reaction mixture contained 65 mM Tris-HCl (pH 7.4), 87.5 PM [1-‘4C]palmitoyl-CoA (2.2 Ci/mol), 100 PM l-palmitoylglycerophosphocholine and protein in 0.5 ml. Lipids were extracted as described above and phosphatidylcholine was separated by TLC in CHC13/CH30H/CH3COOH/H~0 (50 : 35 : 4 : 2, v/v). The reaction was linear with time for 5 min and with protein concentration up to 200 pug/ml. Morphologic analysis. Explants were prepared for light and electron microscopy as described previously [6]. Morphologic changes were quantitated with the electron microscopic preparations. Type II cells were identified by tne presence of lamellar bodies in the cytoplasm and counted. The number of lamellar bodies in the lining cells contiguous with the lumen was also determined. Data were expressed as the number of lamellar bodies or type II cells per 10 alveolar lining cells. Statistical analysis. In all experiments the lungs of fetuses from 2-4 litters were pooled and this same pool of lungs used for both the control and experimental groups. Statistical analysis was by Student’s t-test for dependent variables. Chemicals. Radiochemicals were purchased from New England Nuclear, (Boston, MA). Phospholipid standards were obtained from Serdary Research Laboratories (London, Ontario). All other biochemicals were purchased from Sigma (St. Louis, MO). Results Phospholipid metabolism Explants of both 18 and 19 day fetal rat lung were exposed to estradiol for 24 and 48 h and the rate of incorporation of choline into phosphatidylcholine determined. Although some stimulation was observed at all ages studies, the optimal response was found when 18 day lung was cultured for 48 h. Stimulation of choline incorporation into phosphatidylcholine was observed with 17pestradiol concentrations varying from 10 ~3 M to 10e5 M. The dose-response study was performed twice and the estrogen/control ratios were as follows: 10m8 M, 1.18; 10e7 M, 1.30; 10m6 M, 1.27 and 10V5 M, 1.52. Since maximal stimulation was seen with the highest dose all further experiments were conducted with 18 day explants cultured for 48 h in lo-’ M 17&estradiol. As is shown in Table I, the incorporation of choline into phosphatidylcholine, disaturated phosphatidylcholine, and sphingomyelin was significantly stimulated by exposure to estrogen. There was also a trend towards an increased rate of incorporation into lysophosphatidylcholine. Incubating the explants with

379 TABLE I THE INFLUENCE OF ESTROGEN ON THE INCORPORATION IN FETAL RAT LUNG IN ORGAN CULTURE

OF CHOLINE INTO PHOSPHOLIPID

ExpIants of 18 day lung were cultured for 48 h as described in the text. Estrogen treated cultures were exposed to 10m5 M l’lfi-estradiol. Data are expressed as pm01 choline incorporated per mg protein per h and represent the mean f SE. of 6 experiments. Statistical significance was evaluated by t-test for dependent variables. Phosphohpid

Control

Estrogen

Phosphatidylchohne Disaturated phosphatidylcholine Sphingomyelin Lysophosphatidylcholine

961 281 33.2 34.5

1285 357 49.1 44.2

i 134 f 32 5.2 ? 4.0 r

f 143 + 39 8.6 f 5.4 +

Estrogen Control 1.37 1.28 1.47 1.31

f 0.07 + 0.11 + 0.05 +_0.14

P

co.005 <0.05
TABLE II THE INFLUENCE OF ESTROGEN ON THE INCORPORATION IN FETAL RAT LUNG IN ORGAN CULTURE

OF ACETATE INTO PHOSPHOLIPID

The data are expressed as dpm f3HIacetate incorporated into phosphohpid per mg protein per h (X10’). Each value represents the mean + SE. of 6 experiments. Culture conditions and statistical analysis were as described in Table I. Phosphohpid

Control

Estrogen

Estrogen Control

1.63 1575.0 * 108.8 965.4 + 57.3 Total phosphohpid 1.64 1179.5 * 85.7 717.7 + 42.0 Phosphatidylcho~e 1.60 583.1 +_ 26.7 368.8 + 23.1 Disaturated phosphatidylcho~e 1.52 175.6 t 13.2 116.7 f 8.5 Phosphatidyiethanolamine 8.2 2.11 86.9 + 41.9 f 4.2 Phosphatidylglycerol 5.4 1.44 66.6 f. 46.6 + 4.4 Phosphatidyhnositol plus phosphatidylserine 2.4 1.53 43.6 + 28.7 f 1.5 SphiigomyeIin The other phosphohpids accounted for less than 2% of the phospholipid radioactivity.

+ f f + * +

0.08 0.08 0.12 0.11 0.17 0.09

+ 0.06

P

(0.001
TABLE III THE INFLUENCE OF ESTROGEN ON THE INCORPORATION OF RADIOACTIVITY ACETATE IN PHOSPHOLIPID IN FETAL RAT LUNG IN ORGAN CULTURE

FROM [3H]-

Data are expressed as the percentage of total phospholipid radioactivity derived from [ 3HIaeetate in each phospholipid fraction. Each value is the mean f SE. of 6 experiments. Culture conditions and statistical analysis were as described in Table I. n.s.. not significant. Phosphohpid

Control

PhosphatidyIcho~e 74.4 * 0.5% Disaturated phosp~tidylcho~e 36.4 + 1.0% Phosphatidylethanohmiine 12.2 f 0.4% Phosphatidylghwerol 4.3 l 0.2% Phosphatidyiinositol plus 4.9 * 0.4% phosphatidylserine Sphiigomyebn 3.0 * 0.1% The other phospboIipids accounted for less than 2% of the

Estrogen

Estrogen Control

P

74.8 35.3 11.1 5.5 4.3

1.01 0.97 0.92 1.29 0.88

* 0.01 + 0.06 * 0.04 * 0.07 + 0.02

n.s.

2.8 * 0.2% 0.94 i 0.06 phospholipid radioactivity.

n.s.

+ 0.7% + 1.9% * 0.4% * 0.3% * 0.3%

ILS.

n.s. <0.005 co.005

380

TABLE

IV

THE INFLUENCE OF ESTROGEN ON THE ACTIVITY SIS IN FETAL RAT LUNG IN ORGAN CULTURE

OF ENZYMES

Values reported are the means + SE. of the number of determinations and statistical analysis were as described in Table I. n.s., not significant. E”zVme

Control

Lysophosphatidic acid acyltransferase * Phosphatidate phosphatase * Choline kinase * * Cholinephosphate cytidylyltransferase ** Cholinephosphotransferase ** Lysolecithin acyltransferase * Glycerophosphatc phosphatidyltransferase plus phosphatidylglycerophosphatase

4.9 10.7 697 829 1017 4.7 45.6

* Activity ** Actib-ity

expressed expressed

as nmol/mi” as pmol/min

**

OF PHOSPHOLIPID

in parentheses.

Culture

(7) (5) (5) (5) (5) (7) (3)

5.6 12.1 686 714 1117 4.6 48.3

conditions

P

Estrogen * 0.4 f 1.2 * 49 + 82 + 74 + 0.2 i 3.8

SYNTHE-

+ 0.5 f 1.8 t_ 32 + 74 + 91 t 0.3 + 0.2

(7) (5) (5) (5) (5) (7) (3)

n.s. tl.S.

“.s. n.s. n.s. tl.S.

n.s.

per mg protein. per mg protein.

the ethanol solvent in which the l’lp-estradiol was dissolved had no effect on the rate of choline incorporation into phosphatidylcholine. The incoporation of [3H]acetate into phospholipid was also studied after exposure to estrogen for 48 h. As is shown in Table II, there was significant stimulation of acetate incorporation into every phospholipid fraction examined. Incorporation into total phospholipid was increased by 63%. Incorporation into individual phospholipids was similarly increased, except that into phosphatidylglycerol and phosphatidylinositol plus phosphatidylserine combined which was increased by 111 and 44%, respectively. Since the increased rate of incorporation of both choline and acetate into phospholipid could have been due to a decrease in the pool size of these precursors, the data were also expressed in terms of the distribution of radioactivity from r3H]acetate in the various phospholipid fractions (Table III). When the data were expressed this way it was found that there was a significant increase in the percentage of radioactivity in the phosphatidylglycerol fraction and a significant reduction in radioactivity in the phosphatidylinositol plus phosphatidylserine fraction. There was no change in the percentage of radioactivity in phosphatidylcholine or disaturated phosphatidylcholine. These findings suggest that estrogen specifically enhances the incorporation of acetate into phosphatidylglycerol. Since the incubation was continued for 4 h, it is unlikely that the increased radioactivity in phosphatidylglycerol merely reflects an increased rate of turnover. There was, however, no significant change in the activities of any of the enzymes of phospholipid synthesis that were examined in this study (Table IV). Morphology

There was a tendency towards an increase in the number of lamellar bodies and type II cells in the estrogen treated explants, but the differences were not statistically significant. There were 9.2 + 1.6 lamellar bodies per 10 alveolar lining cells in the control cultures and 12.7 f 2.7 in the esterogen treated explants. The control cultures had 4.7 f 0.5 type II cells per 10 alveolar lining

381 TABLE V THE INFLUENCE

OF ESTROGEN

ON GLYCOGEN

PLANTS OF FETAL RAT LUNG IN ORGAN

CONTENT

AND PROTEIN:DNA

RATIO OF EX-

CULTURE

Values reported are the means of 4 experiments f S.E. Culture conditions and statistical analysis were as described in Table I. n.s., not significant.

Control Estrogen Estrogen:control P

-

Glycogen content

Protein/DNA

Wglmg protein)

@g/l.&

184.3 + 7.4 147.4 + 4.0 0.81 + 0.05 <0.05

6.4 f 0.5 6.0 + 0.3 0.94 i 0.04

cells, whereas the esterogen

treated

ILS.

cultures had 5.7 + 0.7 type II cells.

Glycogen, DNA and protein con tent As is shown in Table V, exposure of the explants to estrogen resulted in a significant reduction in the glycogen content. There was no change in the protein/DNA ratio. Discussion A number of clinical and animal studies have suggested that there may be a relationship between estrogen and fetal lung maturation [16-201. More conclusive evidence was recently provided by Khosla and Rooney [3] who showed that administration of 17@estradiol resulted in a marked increase in the phosphatidylcholine content of fetal rabbit lung lavage. Estradiol treatment also resulted in an increased rate of choline incorporation into phosphatidylcholine in fetal rabbit lung slices [21]. These changes suggest that estrogen stimulates fetal lung maturation and surfactant production. Previous studies have also shown that estrogen stimulates the synthesis of adrenal corticosteroids [5] and the synthesis and secretion of pituitary prolactin [4]. Although the role of prolactin has not been established [ 2,221, corticosteroids have been shown to be potent stimulators of fetal lung maturation [23] and it was possible that estrogen was acting indirectly through corticosteroids or prolactin. In order to established whether estrogen was acting directly on the fetal lung or via other hormones, it was necessary to examine its effect on fetal lung in vitro. Organ culture of fetal lung is a useful system for examining hormonal influences on lung development as the explants can be cultured in chemically defined serum-free medium, remain sensitive to hormonal action, and continue biochemical and morphological development in vitro [6]. Since nutrients, hormones and oxygen reach the cells of the tissue block by diffusing through a one mm thick mass of tissue, high concentrations of these substances are generally required. The concentrations of hormones or other agents used with this system cannot therefore be related to the concentrations which are used in vivo. The data presented here indicate that exposure to estrogen results in a significant increase in the rate of choline and acetate incorporation into a number of phospholipids including the major surfactant components, disaturated phos-

382

phatidylcholine and phosphatidylglycerol. An increase in the apparent rate of incorporation of a radioactive substrate into its end product can be due to a decrease in the intracellular pool of that substrate or to an increase in its rate of turnover. In an attempt to avoid pool size problems, we also expressed the acetate incorporation data in terms of the distribution of radioactivity from [3H]acetate in the various phospholipid fractions. When the results were expressed in this way, it was found that exposure to estrogen resulted in a significant increase in the percentage of radioactivity in the phosphatidylglycerol fraction and a decrease in the phosphatidylinositol plus phosphatidylserine fraction, The percentage of radioactivity in the other phospholipids was virtually unchanged. These data indicate that estrogen acts directly on the fetal lung to stimulate precursor incorporation into phospholipid. It may specifically enhance incorporation into phosphatidylglycerol, the second most abundant phospholipid in pulmonary surfactant. Estradiol receptors have been detected in fetal lung [24]. Like other steroid hormones, estrogen is believed to act by initially binding to a receptor in the cell cytoplasm. The receptor-steroid complex is then translocated to the nucleus where the synthesis of messenger RNA and ultimately enzyme protein is initiated. We were unable to detect a significant change in any of the enzymes of phospholipid synthesis which were examined in this study. This finding was unexpected, since Rooney et al. [25] have found enhanced activity of cholinephosphate cytidylyltransferase, lysolecithin acyltransferase and glycerophosphate phosphatidyltransferase plus phosphatidylglycerophosphatase in fetal rabbit lung after estrogen treatment. The discrepancy may be due to species differences or to differences in the models used in the two studies. Estrogen treatment also resulted in a significant decrease in the glycogen content of the lung. This decrease in glycogen content by a hormone which may also stimulate surfactant synthesis provides further support for the theory that glycogen is a source of substrate for pulmonary phospholipid biosynthesis in the fetus [ 111. Other agents which stimulate lung surfactant production such as corticosteroids and cyclic AMP also procedure a decrease in lung glycogen content [23,26], whereas insulin which may inhibit lung maturation and surfactant production conversely produces an increase in lung glycogen content

[61. The data presented in this paper provide further evidence a role in the regulation of fetal lung maturation and indicate effects by a direct action on the fetal lung.

that estrogen plays that it produces its

Acknowledgements We thank Donna Light for assistance with the electron microscopy and Debra Camputaro for help with the manuscript. This research was supported by U.S.P.H.S. grants no. HL 19752 and HD 10192. References 1

Van

2

Farrell,

Golde. P.M.

L.M.G. and

(1976) Hamosh,

Am.

Rev.

M. (1978)

Resp.

Dis.

Clin.

Perinat.

114.977-1000 5.197-229

383

3 Khosla, S.S. and Rooneu, S.A. (1979) 4 Kanematau,

S. and Sawyer,

C.H. (1963)

5 Kitay, J.I. (1961)

Nature 192, 358-359

6 Gross,

G.J.W..

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

I., Smith,

Maniscalco,

Am. J. Obstet. Endocrinology W.M.,

Czajka,

Gynecol.

133, 213-216

72,243-252 M.R.,

Wilson,

C.M. and Rooney,

S.A. (1978)

J.

Appl. Physiol. 45. 355-362 Bhgh, E.C. and Dyer, W.J. (1959) Can. J. Biochem. Physiol. 37, 911-917 Rooney, S.A., Nardone, L.L., Shapiro, D.L.. Motoyama, E.K. and Gobran. L. (1977) Lipids 12, 438442 Mason. R.J., NeIIenbogen, J. and Clements, J.A. (1976) J. Lipid Res. 17. 281-284 Lowry, O.H., Rosebrough. N.J.. Farr, A.L. and Randall. R.J. (1951) J. Biol. Chem. 193. 265-275 Maniscalo, W.M., Wilson. C.M., Gross, I., Gobran, L.. Roomy, S.A. and Warshaw, J.B. (1978) Biochim. Biophys. Acta 530,333-346 Hill, B.T. and Whatley, S. (1975) FEBS Lett. 56, 20-23 Rooney. S.A.. Wai-Lee, T.S.. Gobran, L. and Motoyama. E.K. (1976) Biochim. Biophys. Acta 431, 447-458 Roomy, S.A. and Wai-Lee, T.S. (1977) Lung 154, 201-211 Rooney, S.A., Gross. I., Gassenheimer, L.N. and Motoyama, E.K. (1975) Biochim. Biophys. Acta 398, 433--441 Dickey, R.P. and Robertson, A.F. (1969) Am. J. Obstet. Gynecol. 104, 551--555 Conly. P.W., LeMaire, W.J., Monkus, E.F. and Cleveland. W.W. (1973) J. Pediatr. 83, 851-m853 Shankhn, D.R. and Wolfson. S.L. (1970) J. Reprod. Med. 5, 53-72 Spellacy. W.N., Buhi, W.C., R&al, F.C. and Holsinger, K.L. (1973) Am. J. Obstet. Gynecol. 115, 216-219 Abdul-Karim, R.W.. Prior, J.T. and Havilland, M.E. (1969) J. Reprod. Med. 2,140-146 Khosla, S.S. and Rooney, S.A. (1978) Physiologist 21,64 Ballard. P.L., Gluckman, P.D., Brehier, A., Kitterman, J.A.. Kaplan, S.L., Rudolph, A.M. and Grumbath, M.M. (1978) J. CIin. Invest. 62. 879-883 Rooney, S.A., Gobran, L.I., Marino, P.A., Maniscalco. W.M. and Gross, I. (1979) Biochim. Biophys. Acta 572, 64.-76 Pasquahni. J.R., Sumida. C.. GelIy. C. and Nguyen, B.L. (1976) J. Steroid Biochem. 7, 1031-1038 Rooney, S.A., Gobran, L.I. and Khosla, S.S. (1979) Fed. Proc. 38, 235 Maniscalo. W.M., Wilson, C.M. and Gross, I. (1979) Pediatr. Res., in the press