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Sex-specific differences in rabbit fetal lung maturation in response to cpidermal growth factor Jonathan M. Kleinand Heber C. Nielsen B~t,~J P,','ouatd Cvm¢'r, D¢,~mmo~r ~ f Ped~¢Jtrh ~, .%'~,L~p£ .~[~ml ,~h'~tit"M ~'em,'r. ,q~.ffr t. IrA (U.S.4.)

(Rcccked "i March Iqtl] ) (l~evised rnanu,~crlpt rccciw'C 15 Jill? [ qq l )

Key words: Epidermal growth factor: Fetal bung rn~turzlti~.Jix:~cx-.;flccifi(.'; Surfaciant

Males and females exhibit different stages at" lung development at the same gestation with mates lagging behind. We hypothesized that one of the mechanisms responsible for the sex-speclfle difference in fetal tong 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 cabblt lung explants prepared at 21 and 24 days gestation (term 31 days). The explants were maintained in Waymouth's media + I0% stripped fetal calf serum with or without EGF (10 n g / m l ) . The incorporation of [ 1,3-t4C]glycerol into l a m d l a r body DSPC was assessed after 3, $, or 7 days of culture. Female lung explants prepared at 21 days of gestation had increased incorporation nf glyoerol into DSFC over time in response to EGF treatment. Male Inn~t explants preparett at 21 days did not respond to EGF treatment, in explants prepared at 24 days gestation, baselitte glycerol incorporation into DSPC was higher in female as compared to male fetal lung explants, EGF-respnnsiveness 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 nf the mechanisms responsible for the lag in male fetal lung development is a delay in the onset of EGF activity.

Introduction

Epidermal Growth Factor (EGF) is one of a number of biologically active extraceliular signals involved in the regulation of growth, development and differentiation [1]. EGF is a poiypeptide hormone consisting of a single chain of 53 amino acid residues with a molecular weight of 6045 Da ill. E G F has been shown to enhance the differentiation of the fetal lung in several ways. First, acute stimulation of fetal rat lung organ cultures with E G F causes an increase in phosphatidylcholine (PC) synthesis [2], Second, culturing fetal [ung [throblasts in the presence of EGF leads to accelerated production of fibroblast pneumoaocyte factor (FPF), a factor that induces the differentiation of type 11 cells

Correspondence: J.M. Klein, Deparlrn*nt o[ Pediatrics. Universit~ of Iowa, Itr~a City, IA 52242, U.$,Pt,

[3]. Both of these effects of EGF are dependent on the gestational age of the. total tung tissue [2,3], Third+ in cultured human fetal lung tissue. EGF enhances the synthesis of the surfactant-assoclated protein SP-A [4]. Fourth, EGF given in viva accelerated fetal rabbit lung maturation in terms of morphology, compliance and surfactant phospholipid synthesis [5,6]. Surfactant is produced in the tung by differentiated type II cells and is stored intracellularly in lamellar bodies [7]. Previous studies have shown a sex-specific difference in fetal hmg maturation characterized by surfactant synthesis occurring earlier in females than in males [8]. We hypoIhcsizcd that a ~cx-specific difference in the effects of EGF on fetal lung development is one of the factors responsible for the delay in fetal lung maturation of the male. To test this hypothesis, w~ cwuuateu the production of lamellar body phospholipids in the presence or absence of EGF in sex-specific fetal rabbit lung explants prepared at different gestational ages.

122 Materials and Methods

h in order to study the synthesis of surfactant F,,,ospholipids.

Animals Pregnant New Zealand White rabbits of known gestation (day of m a t i n g ~ day 0; term ~ ql days, Pine Acres Ra~,bitry, West Brattleboro, VT) were sacrificed with an intravenou~ injection of sodium pentobarbital (6(10 rag) on days 21 and 24 of gestation, The expert, ment was repeated four times using 12 litters per ex'per;ment at day 21 and 4 litters per experiment at day 24 of gestation. The uteri were removed and the fetuses were collected under sterile conditions. The sex of each fetus was identified [9] and the lungs were removed and pooled by sex in serum-free Waymouth's MB 752/1 medium with L-glutamine (Gibco Laborato, ries, Grand Island, NY). The medium also contained NaHCO 3 2.5 m g / m l , aqueous penicillin G I(10 U / m l , streptomycin 100 p g / m I and amphoter~ein 2.5/zg/ml,

Organ culture Sex specific cultures of the fetal htngs were prepared as previously described [g,10]. The pooled lungs were minced into 1 mm 3 pieces with a sterile razor blade. The minced tissue was then placed on a piece of sterile lens paper, which was resting on three-metal grids inside of a 20 ~' 100 mm culture dish containing 9 ml of Waymouth's medium with 10% charcoal-strlpped fetal calf serum. Charcoal stripping was accomplished by adding activated charcoal (1.0 g) and Dextran T-70 (0.1 g) (Sigma Chemica[ Company, St. Louis, M e ) to 10 ml of phosphate buffered saline, which was then stirred for 1 h on ice. The mixture was centrifuged (1200 ×g for 10 rain), the supernatant was discarded and 200 ml of fetal calf serum was added to the pellet. The calf s e r u m / c h a r c o a l pellet mixture was stirred on ice for ] b and centrifuged (1200 × g for 10 rain). The supernataut was mixed with a second charcoal/dextran pellet thal had been prepared as described above, stirred for 1 h on ice and centrifuged (1200 × g for 10 rain). The supcrnatant was filtered through a 0.45 # m filter until the serum was clear. The efficicticy of EGF removal by charcoal stripping was evaluated by adding 2 ng of [t-*5I]EGF (specific activity 100 /.tCi/¢zg) to 10 ml of fetal calf serum, a sample reference aliquot was removed and the remaining fetal calf serum was charcoal stripped by the above method. A 1 ml aliquot of the stripped serum was colinted and the amount of EGF removed by stripping was expressed as a percentage of the counts initially added. Fresh EGF (Sigma) (10 n g / m l ) and media were added daiIy to the explaats which were maintained at 3 T C in a humidified atmosphere of 5% CO z and 95% air for 3, 5, or 7 days, At the end of the culture period, [ 1,3- ~4C]glycerol (I # C i / m l , specific activity 50 m C i / m m o l ; ICN Radiochemicals, Irvine, CA) was added to the explants for 24

Lamellar body isolation Lamellar bodies were isolated in order to evaluate surfactant phospholipid synthesis [11]. At the end of the incubation period, the fetal lung explants were homogenized in 5 ml of lamellar body buffer (0.15 M NaCI, 0.1 mM EGTA, 50 mM Tris, pH 7.40) at 4~C. Protein concentration was measured in 5 ¢zl aliquots of the homogenate [12]. The lamellar bodies were isolated from the remaining homogenatc as described previously by differential centrifugation using discontinuous sucrose density gradients [11]. The isolated lamellar body pcllcts were stored at -70°C until subsequent phespholipid analysls~ In previous studies using sex-specific fetaE lung organ cultures we have found that the efficiency of lamelIar body recovery in males was slightly higher than females (males 36 + 0.5%, females 32 + 1.0%, mean + S.D., P < 0.05) [8].

Phospholipid analysis The lamellar body pelIets were suspended in normal saline and the lipids were extracted with chloroform and methanol [13]. An aliquot of the extract was combined with osmium tetroxide to assay disaturated phosphatldyleholine [1,11, which was identified using thin layer chromatography on silica gel plates (Kodak Chromagram Sheet/13179; Eastman Kodak Company, Rochester, NY) in chloroform : methanol:water, 65 : 35:4 [15], The lipid spots were scraped into vials and the amount of incorporated [~4C]glycerol was measured in a scintillation counter and expressed in dpm per mg of homogenate protein.

Statistical analysis The significance of the effects of sex, time in culture, gestational age and EOF treatment were evaluated by two-way analysis of variance, Interactive effects of sex, treatment and time were a h n evaluated to test specifically whether the responses to E G F treatment were different by sex [161. Values were considered statistically significant if P values were less than 0.05. Values were expressed as the mean ± S.E. of four experiments performed at each gestational age. Results In fetal lung cxplants prepared at day 21 of gestation, there was no significant difference in glyceroi incorporation between control male and female ex+ plants. FurthcrmoJe, there was no significant change in the rate of incorporation of [;4C]glycerol into DSPC with the number of days in culture in either male or female control explants (Fig. 1A and 1B). However,

t23 200

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rate ~f glycorul incorp~ration into DSPC as compared to male explants (Fig. 2A and 2B, P - 0.OOl). Second, EGF responsiveness as measured by DSPC synthesis w a s s i g , q f i c a n l l y different when comparing males to females (P = II.(Ig). In the cultures prepared at day 24 of gestation, fhc difference was characterized by a consislent stimulation of DSPC synthesis i,a male explains and bv a minimal decrease in DSPC synthesis in female explants (Fig. 2). in mate explants, the rate of glycerol incorporation into DSt'C was increased with EGF trea~mcm by 55% after 3 days. by 76% after 5 days trod by 27c÷ after 7 days (Fig. 2B). These increase~ in DSPC s y n l b e s i s in males treated with EGF were sufficient to bring their level of synthesis to the control female level In cortlrast, the rate of glycerol incorporation into DSPC in female cultures treated with EGF

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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 mechan...
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