Main

Phosphatidylcholine is actively synthesized de novo in the fetal lung and comprises over seventy percent of the lipid portion of pulmonary surfactant. DSPC is the major surface-active lipid component of surfactant that is responsible for maintaining stability of the alveoli (1). A deficiency of pulmonary surfactant is the primary cause of neonatal RDS, characterized by diffuse atelectasis, ventilatory impairment, and gas-exchange abnormalities. Previously, it has been shown that male infants are at greater risk for developing RDS than female infants of the same gestational age, and male mortality from this disorder is nearly twice that of females (2). Glucocorticoids, which are used to prevent RDS, also seem to be more effective in females than in males (3). Additional work has revealed significant gender differences in fetal lung maturation and surfactant phospholipid content in the human, rabbit, and rodent (4–8).

Sex-related differences in surfactant content have led to studies investigating whether these observations may be due to differences in the biosynthesis of phospholipid. Indeed, prior studies in the fetal rabbit have suggested that the late gestational surge in phosphatidylcholine production is not only delayed in males compared with females but also is sluggish in the male in response to treatment with stimulators of surfactant lipid synthesis such as dexamethasone and epidermal growth factor (9, 10). Male deficiencies in surfactant synthesis also seem to have been identified in the rat model (11–14). Torday and Dow (11) showed that female lung cells synthesize more surfactant lipid compared with male lung cells and that these differences can be abolished by glucocorticoid and thyroid pretreatment. Other studies showed sex-specific differences in the release of a soluble factor from fibroblasts, which is stimulatory for surfactant production (12). These differences were primarily seen in response to sex hormones (13). Collectively, these studies suggest that differences in surfactant content between male and female fetuses may be attributed to gender differences in surfactant phosphatidylcholine synthesis.

The principle pathway in mammalian tissues for the biosynthesis of phosphatidylcholine is the CDP-choline pathway. The sequential steps in this pathway involve cellular uptake of choline, choline phosphorylation by CK (EC 2.7.1.32), conversion of choline phosphate to CDP-choline by CT (EC 2.7.7.15), and finally generation of phosphatidylcholine by CPT (EC 2.7.8.2). A consistent feature among essentially all studies to date examining gender differences in phosphatidylcholine metabolism is that surfactant production was assessed primarily by determining the incorporation of a radiolabeled precursor such as choline into the product, phosphatidylcholine (or DSPC). However, differences in incorporation rates of radiolabeled precursors into phosphatidylcholine could be regulated at several steps, such as cellular choline transport, limitations of pool sizes of choline substrates, and enzymatic sites within the CDP-choline pathway. Further, to date, the possibility that gender differences in phospholipid content may be secondary to altered DSPC catabolism between the sexes has been ignored. Herein, we show that sex-specific differences in surfactant phospholipid content are due, at least in part, to differences in choline transport and the activity of CT, the rate-limiting enzyme for surfactant phosphatidylcholine synthesis (15). However, we observed no sex-specific differences in DSPC turnover.

METHODS

Materials.

The choline, CK, choline phosphate, cortisol, and lipids including phospholipid standards were purchased from Sigma Chemical Co. (St. Louis, MO). Polyclonal TGF-β antibody and TGF-β were purchased from R & D Systems (Minneapolis, MN). Anion exchange resin (AG1-X8, formate form) was obtained from Bio-Rad (Hercules, CA). Silica LK5D (250 mm × 20 × 20 cm) TLC plates were purchased from Whatman International (Maidstone, England). Waymouth's medium, MEM, and choline-free medium were obtained from the University of Iowa Tissue Culture and Hybridoma Facility (Iowa City, IA). Cell numbers were determined using a Coulter Z1 Dual Particle Counter (Coulter Corp., Miami, FL). All radiochemicals were purchased from DuPont New England Nuclear Chemicals (Boston, MA).

Animals and cell preparation.

Timed pregnant Sprague-Dawley rats at d 20 gestational age (d = 0 designated by presence of vaginal sperm plug) were obtained from Harlan Sprague-Dawley Inc. (Indianapolis, IN). After deep anesthesia with phenobarbital (50 mg/kg i.p.), fetal rats were delivered by cesarean section from their dams. These procedures were approved by the University of Iowa Animal Care and Use Committee. The fetal rats were sexed by microscopic detection of gonads and confirmed in some studies histologically (16). The male and female lungs were resected, separately pooled, homogenized, and used directly for biochemical studies. In separate studies, pooled tissues were placed in calcium and magnesium-free Hanks' balanced salt solution and subsequently used to isolate male and female mixed monolayer cultures (17). Briefly, lungs were minced into ∼1-mm pieces in Hanks' balanced salt solution before treatment with trypsin 0.05% and 10 μg/mL DNAse to disperse cells. After the cell suspension was gravity filtered through a 50-μm Nitex filter, the filtrate was centrifuged to obtain a pellet, and the pellet was suspended in Waymouth's medium containing 10% CS-FCS. The harvested cells were plated to allow for differential adherence of some fibroblasts as described (17). The nonadherent cells were carefully removed, pelleted, sonicated at 4°C, and resuspended in buffer A [150 mM NaCl, 50 mM Tris, 1.0 mM EDTA, 2 mM DTT, 0.025% sodium azide, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4] and used directly for enzyme analysis. Nonadherent cells were also plated at a density of 8 × 106 cells per 100 mm tissue culture dish in Waymouth's medium containing 10% CS-FCS and cultured for various times. In subsequent studies, the nonadherent cells consisting of mixed lung cells underwent an additional 1-h differential adherence step to allow for removal of fibroblasts (17). Confluent attached fibroblast monolayers were rinsed with MEM and replaced with fresh medium alone, medium containing cortisol [1 × 10−7 M (17)], polyclonal TGF-β antibody [10 μg/mL (17)], or the combination of cortisol (1 × 10−7 M) plus polyclonal TGF-β antibody (10 μg/mL) for 24 h. The medium from fibroblasts under these conditions was aspirated from cultures, centrifuged to remove cell debris, and stored at −70°C. The nonadherent male and female fetal alveolar pretype II epithelial cells isolated from the second adherence step were plated in Waymouth's medium containing 10% CS-FCS for 48 h. The medium was then removed and replaced with either control medium alone or fibroblast-conditioned medium containing cortisol, polyclonal TGF-β antibody, or cortisol-conditioned medium plus polyclonal TGF-β antibody. Before addition to pretype II cells, each fibroblast-conditioned medium was mixed 1:1 with MEM. After 24 h of culture, cells were rinsed and harvested. Finally, in some studies, TGF-β [10 ng/mL (17)] was added directly to pretype II cells.

Phosphatidylcholine metabolism.

Phosphatidylcholine content was determined after cellular lipid extraction, separation of phospholipids by use of TLC, and quantitative analysis by the phosphorus assay (18). DSPC synthesis was assessed by pulsing cells with 2 μCi of [methyl-3H]-choline chloride for the final 4 h of incubation, and levels of DSPC were determined using osmium tetroxide as described previously (18). DSPC degradation was determined by rinsing cells in choline-free medium three times and pulsing cells overnight with 5 μCi of [methyl-3H]-choline chloride per dish. Cells were then rinsed the next day and incubated with Waymouth's medium containing 500 mg/mL choline. After various times, cells were processed for analysis of DSPC as described above.

Enzymes of phosphatidylcholine hydrolysis.

PLA2 activity was assessed by determining the release of [3H]-arachidonic or [3H]-palmitic acid into medium after the cells were pulsed for 1 h with 1 μCi per dish of label (19). The cells were rinsed three times after the pulse with Waymouth's medium containing 0.1% fatty acid-free albumin and subsequently incubated for various times periods. Aliquots of medium were taken for scintillation counting and corrected for total counts in each dish. In addition, direct exogenous assays for acidic calcium-independent PLA2 and PC-PLC were conducted (20). The reactions were linear up to 500 μg of protein in the reaction mixture. PLD activity was measured by assaying transphosphatidylation activity of PLD in the presence of ethanol exactly as described (21).

Enzymes of the CDP-choline pathway.

The activity of CK was assayed as described (22). The reaction mixture (0.1 mL volume) contained 100 mM Tris/HCl buffer (pH 8.0), 10 mM Mg acetate, 0.016 mM [14C]-choline (specific activity, 0.32 μCi/mM), 10 mM ATP, and 40 μL of protein. After a 1-h incubation at 37°C, the reaction was terminated with 0.02 mL of cold 50% trichloroacetic acid. After drying, aliquots of the mixture were spotted on Whatman 3MM paper, and choline metabolites were resolved as described (22). The spots that comigrated with radiolabeled standard choline phosphate were cut and used for scintillation counting. The reaction was linear with time up to 60 min and up to 200 μg of cellular protein in the incubation mixture. The recovery of the product, [14C]- choline phosphate, was 78%.

The activity of CT was determined by measuring the rate of incorporation of [methyl-14C]phosphocholine into CDP-choline by using a charcoal extraction method (18, 23). The reaction mixture and optimal assay conditions have been described previously (18). Unless stated otherwise, all assays were performed without the inclusion of a lipid activator in the reaction mixture.

The activity of CPT was assayed as described (24). Each reaction mixture contained 50 mM Tris-HCl buffer (pH 8.2), 0.1 mg/mL Tween 20, 1 mM 1,2-dioleoylglycerol, 0.8 mM phosphatidylglycerol, 0.5 mM mM [14C]-CDP-choline (specific activity, 0.50 μCi/μM), 5 mM DTT, 5 mM EDTA, 10 mM MgCl2, and 40 μL of enzyme. The lipid substrate was prepared by combining appropriate amounts of stock solutions of 1,2-dioleoylglycerol and phosphatidylglycerol in a test tube, drying under nitrogen gas, and brief sonication before addition to the assay mixture to achieve the final desired concentration. The reaction proceeded for 1 h at 37°C and terminated with 4 mL of methanol:chloroform:water (2:1:7, vol:vol). The remainder of the assay was performed exactly as described (24). The reaction was linear with time up to 60 min and up to 100 μg of cellular protein in the assay mixture.

Choline transport.

Choline transport was assessed by rinsing cells in choline-free medium three times and preincubating the cells in this medium for 1 h. Cells were then pulsed for 15 min with [methyl-3H]-choline chloride at 37°C, after which the medium was discarded, and the cells were rinsed again and chased in choline-free medium alone for an additional 1 h (25, 26). Cells were scraped in methanol:water (5:4, vol:vol). After organic solvent extraction of lipids, the aqueous phase was dried, and aliquots of the mixture were spotted on 2 × 9-cm strips of Whatman 3MM paper and the choline metabolites were resolved in ethanol:ammonia:isopropanol [65:35:20 vol: vol (26)]. The areas corresponding to choline and choline-phosphate standard were cut and placed into 10 mL of scintillation fluid for counting.

Choline pool size.

Choline mass was assayed as a modification of the enzymatic procedure described by Post et al. (15). Each reaction contained 88 μL of protein residue, 100 mM glycylglycine (pH 9.2), 4 mM MgCl2, 6 mM ATP, 4 μCi (γ-32P)ATP, and 0.1 U CK in a final volume of 200 μL. The reaction was terminated with the addition of 200 μL of cold ethanol after 1 h at 37°C, and the mixture was added to a 1 × 6-cm column prefilled with AG1-X8 resin. The choline phosphate product was eluted with three 1-mL volumes of 0.1 M ammonium bicarbonate, and the resulting effluent was dried and separated from unreacted choline by using TLC as described above. The radioactivity in choline phosphate was compared with a standard curve of choline to determine choline mass. Choline phosphate mass was measured by converting this intermediate to choline by using alkaline phosphatase and subsequent analysis of choline as described above (15).

Protein analysis.

Protein concentration was measured with the Bradford method with BSA used as the protein standard (27).

Statistical analysis.

The data are expressed as the mean ± SEM. Statistical analysis was performed using the t test or ANOVA with a Bonferroni adjustment for multiple comparisons.

RESULTS

Phosphatidylcholine metabolism.

Male and female mixed lung cells were initially isolated and analyzed for DSPC content. Consistent with prior studies, female isolates contained a 53% higher content of DSPC compared with male cells [data not shown (7, 8)]. To investigate the possibility that these quantitative differences in surfactant lipid were due to higher rates of phosphatidylcholine degradation, we performed pulse-chase studies. Cells were pulsed overnight in choline-free medium and subsequently rinsed and incubated in medium before analysis the next day for DSPC at various time points. Under these conditions, there were no significant differences in DSPC detected between the sexes from 2 to 10 h of analysis (Fig. 1A). However, because our labeling was long-term, these studies do not exclude the possibility that there may be different pool sizes of DSPC that exhibit differential rates of turnover within or between the groups. Thus, we performed additional studies directly assaying phospholipase activities. We first measured the release of [3H]-arachidonic acid into the medium after a 1-h pulse (Fig. 2A). Indeed, males exhibited significantly greater release of the fatty acid compared with females over a broad range of time points, suggesting that males have greater cytosolic or secretory PLA2 activity (p< 0.05). However, when labeling was performed with [3H]-palmitate, no substantial sex differences were observed (Fig. 2B). These latter results suggest that there are no sex differences in the activity of PLA2 subtypes that are selectively involved in DSPC degradation. Further, as shown in Table 1, direct exogenous substrate assays revealed there were no significant gender differences in the activities of PLA2, PLD, or PC-PLC after short-term culture. Additional studies performed at other time points of culture also did not reveal differences in the activity of these phospholipases (data not shown).

Figure 1
figure 1

Metabolism of DSPC in male and female fetal lung cells. A, Pulse-chase studies showing rates of DSPC degradation in male and female mixed monolayer cultures. Lung cells were isolated from male and female rat fetuses (d 20 gestation) and cultured separately for 2 d in Waymouth's medium containing 10% CS-FCS. The cells were then pulsed overnight with 2 μCi of [methyl-3H]-choline chloride in choline-free medium. After the cells were rinsed, the levels of DSPC were measured at various time points as described in “Methods.”B, Male and female mixed monolayer cells were cultured for 3 d in Waymouth's medium containing 10% CS-FCS. The cells were then incubated for the final 4 h with 2 μCi of [methyl-3H]-choline chloride. The rates of [methyl-3H]-choline incorporation into DSPC in cells were then measured. The values are expressed as cpm/mg of protein and as the mean ± SEM. The data are representative of three independent experiments. Statistical analysis was performed using the t test. ★p= 0.06.

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Figure 2
figure 2

Phosphatidylcholine hydrolysis in male and female fetal lung cells. PLA2 activity was assessed in male and female mixed monolayer cultures by assaying release of [3H]-arachidonic acid (A) or [3H]-palmitic acid (B) into medium at various time points after pulsing cells for 1 h with 1 μCi per dish of label. The [3H]-fatty acid values are expressed as a ratio of cpm (cpm in medium − cpm blank/homogenate cpm × 1000). The above results are representative of three independent experiments (mean ± SEM). ★The values at each time point between males and females for [3H]-arachidonic acid release were significantly different (p< 0.05).

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Table 1 Activity of phospholipases in male and female fetal lung cells Mixed lung cells were isolated from male and female fetal rat lungs from pregnant rats (d 20 gestation). The activity of enzymes involved in phosphatidylcholine hydrolysis were assayed after 3 d of culture in Waymouth's medium containing 10% CS-FCS. The results represent data obtained from three separate experiments. Each experiment consisted of male and female fetuses obtained from two litters. The activities for PLA2 and phospholipase C are expressed as pmol/h/mg cellular protein. The activity of PLD was measured using the transphosphatidylation activity of the enzyme by incubating cells in the presence of ethanol and assaying the levels of phosphatidylethanol by using TLC (20). The activity of PLD is expressed as cpm recovered in phosphatidylethanol relative to total phospholipid [(cpm recovered in phosphatidylethanol/total phospholipid cpm) × 105].
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In addition to DSPC degradation, analysis was performed to determine choline incorporation into DSPC as a crude measure of surfactant biosynthesis by pulsing cells the last 4 h of incubation. Indeed, mixed lung cell monolayers from females had a 71% greater rate of choline incorporation into DSPC after culturing cells for 3 d (Fig. 1B). Together, these studies suggest that males synthesize less surfactant lipid compared with females; overall, these results do not support substantial differences in DSPC turnover between male and female lung cells.

Enzymes of the CDP-choline pathway.

To confirm sex differences in surfactant lipid synthesis, we assayed the activities of enzymes within the CDP-choline pathway. Assays were performed in freshly isolated mixed lung cells from separately pooled male and female fetuses (Table 2) and after culturing the cells for 3 d (Table 3). Enzyme analysis was also performed using lungs obtained from each individual fetus (Table 4). Finally, enzyme studies were conducted in alveolar pretype II epithelial cells (Table 5). When analysis was performed on freshly isolated mixed lung cells, female cells had 44% greater activity of CT compared with males (p< 0.05). There were no significant differences between the groups with regard to CK or CPT activities (Table 2). After 3 d of cell culture, females comparatively also had modest yet significantly higher activity for CT (Table 3). Analysis of enzyme activities in whole lung homogenates obtained from each individual male and female fetus did not reveal substantial sex-specific differences (Table 4). These results indicate that diminished surfactant lipid synthesis in males relative to females in mixed lung cell culture is due, at least partly, to delayed conversion of choline phosphate to CDP-choline. Additional studies revealed that CT activity in freshly isolated male and female pretype II cells was 197 ± 62 and 207 ± 66 pmol·min−1·mg−1 protein, respectively (n= 2). In male and female pretype II cells cultured for 72 h, there were also no significant differences in CT activity (Table 5), or in CK or CPT activities (data not shown, n= 3). Further, because sex-specific differences in CT activity were not observed in purified pretype II cell isolates, the results suggest that factors present in a mixed-cell system seem to be important in differentially modulating enzyme activity in male and female lung cells.

Table 2 Enzymes of the CDP-choline pathway in freshly isolated male and female fetal lung cells Mixed lung cells from male and female fetal (d 20 gestation) rat lungs were isolated and assayed directly for activity of enzymes involved in phosphatidylcholine synthesis. The results represent data obtained from four separate experiments. Each experiment consisted of male and female fetuses obtained from two litters. The activities for each enzyme are expressed as pmol/min/mg cellular protein.
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Table 3 Enzymes of the CDP-choline pathway in cultured male and female fetal lung cells Mixed lung cells were isolated from male and female fetal rat lungs from pregnant rats (d 20 gestation). The activity of enzymes involved in phosphatidylcholine synthesis were assayed after 3 d of culture in Waymouth's medium containing 10% CS-FCS. The results represent data obtained from four separate experiments. Each experiment consisted of male and female fetuses obtained from two litters. The activities for each enzyme are expressed as pmol/min/mg cellular protein.
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Table 4 Enzymes of the CDP-choline pathway in male and female lungs Individual male and female fetal rat lungs were isolated from pregnant rats (d 20 gestation). The activity of enzymes involved in phosphatidylcholine synthesis were directly assayed in each lung homogenate preparation from each individual fetus. The results represent data obtained from seven separate male and female fetuses. The activities for each enzyme are expressed as pmol/min/mg cellular protein.
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Table 5 Effect of fibroblast-conditioned medium on CT activity in male and female pretype II cells Male and female pretype II alveolar epithelial cells were isolated from pregnant rats (d 20 gestation). Fetal fibroblasts were cultured with cortisol (1 × 10−7 M) in the presence or absence of TGF-β–neutralizing antibody (10 μg/mL) for 24 h. Male and female pretype II cells were subsequently cultured for 24 h in the presence of fibroblast-conditioned medium exposed to cortisol with or without TGF-β–neutralizing antibody. The activity of CT was then assayed. The results represent data obtained from three separate experiments. Each experiment consisted of male and female fetuses obtained from two litters. The activities for enzyme activity are expressed as pmol/min/mg cellular protein. p< 0.001, d vs a or c;p< 0.05, d vs e;p< 0.05, a vs b. Statistical analysis was performed using ANOVA.
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Choline transport.

The differences in choline incorporation into DSPC observed between the sexes could also be attributed to an increase in choline transport or smaller intracellular pools of unlabeled substrates for DSPC, such as choline, in females compared with males (15, 28). To address these possibilities, we first pulsed the cells with [3H]choline in the presence of choline-free medium for 15 min and subsequently chased the cells in the absence of label in the same medium for 1 h. The cellular content of [3H]choline and incorporation into [3H]choline phosphate were then measured (Fig. 3). Mixed lung cells analyzed shortly after isolation (d 1) did not exhibit substantial gender differences in either [3H]choline or [3H]choline phosphate. However, after culture of cells for 3 d, female cells showed a 36% higher rate of cellular [3H]choline transport compared with male cells (p< 0.05, Fig. 3A). When similar analysis was performed in pretype II cells, male and female cells did not differ with regard to rates of choline transport [368 ± 48 versus 370 ± 87 cpm/mg protein, respectively (n= 3)]. The observation that the radiolabeled incorporation of [3H]choline into [3H]choline phosphate was not significantly greater in females compared with males also suggests that the females did not have a smaller pool size of unlabeled choline precursor (Fig. 3B).

Figure 3
figure 3

Choline transport and choline phosphorylation in sex-specific lung cell cultures. Lung cells were isolated from male and female rat fetuses (d 20 gestation) and cultured separately for up to 3 d in Waymouth's medium containing 10% CS-FCS. The cells were pulsed for 15 min with 2 μCi of [methyl-3H]-choline chloride in choline-free medium. The cells were rinsed, and choline metabolites were extracted and processed using TLC for determination of cellular choline uptake and choline phosphorylation as described in “Methods.” The values are expressed as cpm/mg of protein and as the mean ± SEM. The data are representative of three independent experiments. Statistical analysis was performed using the t test. ★p< 0.05.

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Choline pool sizes.

To confirm whether there were sex differences in the pool size of choline substrates, we directly assayed the concentration of choline in freshly isolated and cultured male and female lung cells. In four separate studies, we did not detect significant differences between males and females either in the mass of choline or choline phosphate after initial cell isolation or extended culture (Fig. 4).

Figure 4
figure 4

Choline and choline phosphate pool sizes. Freshly isolated male and female lung cells (d 1) or sex-specific mixed lung cell cultures (3 d of culture) were assayed for choline and choline phosphate content as described in “Methods.” The values are expressed as nmol/mg protein. The results are representative of four independent experiments, and the data are expressed as mean ± SEM.

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Effect of fibroblast-conditioned medium on CT activity.

Because we observed sex-specific differences in phosphatidylcholine synthesis in mixed lung cells but not in purified fetal pretype II cells, we hypothesized that fibroblasts present in the mixed-cell system may release soluble factors that regulate CT activity differently between the sexes. FPF is an important product released from fibroblasts that stimulates surfactant synthesis (12, 13). Corticosteroids trigger the release of FPF, whereas other factors such as TGF-β are inhibitory for FPF-mediated surfactant synthesis (13, 29). In addition, corticosteroids regulate TGF-β expression in fibroblasts (30, 31). To test our hypothesis, fibroblasts were cultured with cortisol, TGF-β–neutralizing antibody, or the combination of cortisol and TGF-β antibody. The fibroblast-conditioned medium was transferred to primary isolates of male and female pretype II cells, and enzyme activity was measured (Table 5). Compared with control, incubation of male cells with cortisol-conditioned medium stimulated CT activity by 41% (p< 0.05, n= 3), but its effect on the female enzyme was modest and did not reach significance. Fibroblast medium treated with TGF-β antibody alone did not alter enzyme activity (Table 5). However, the greatest increase in enzyme activity over control was observed when male and female cells were incubated with fibroblast medium that was preconditioned with both cortisol and TGF-β–neutralizing antibody. Under these conditions, male pretype II cells had significantly greater CT activity compared with female cells treated similarly (Table 5). Thus, under hormonally stimulated conditions, these data suggest that TGF-β serves as a negative regulator of CT activity in male and female lung cells.

DISCUSSION

Prior studies have shown a female advantage in surfactant synthesis in the rodent model that ranges from a modest 25% to a 2-fold higher level compared with gestationally matched male fetuses (11–14). Several mechanisms have been put forth to explain these observations, including delayed surfactant production by the male lung in response to inhibitory testes-derived hormones (8, 10, 13, 14, 32, 33) or a diminished response of the male to corticosteroids, which stimulate surfactant synthesis (3, 10, 12). Many of these studies used a mixed lung cell model to preserve vital epithelial-fibroblast interactions (9–12). These interactions are important because diminished release of stimulatory factors for surfactant synthesis, such as FPF, from male fetal fibroblasts to the male fetal pretype II cell seems to be at least partly responsible for the female advantage (12, 13, 34, 35). Using this model, we investigated potential biochemical mechanisms that could explain differences in incorporation rates of radiolabeled precursors into DSPC shown previously (11–14). We observed for the first time that female fetal lung cells exhibited higher rates of choline transport and greater activity of CT, but there were no sex-specific differences in DSPC turnover. Interestingly, these sex-specific differences in surfactant lipid synthesis were abolished in purified cultures of male and female pretype II cells. However, exposure of pretype II cells to fibroblast-conditioned medium treated with cortisol and TGF-β–neutralizing antiserum produced a greater increase in CT activity in males compared with females. Thus, we conclude that greater amounts of surfactant phospholipid in the female fetus are attributed to greater biosynthetic activity rather than altered phospholipid degradation. In addition, sex differences in CT function in pretype II cells may be attributed, in part, to soluble factors such as TGF-β secreted by fibroblasts in the setting of corticosteroid exposure.

The possibility that gender differences in DSPC content could be secondary to altered degradation of DSPC has not been previously investigated. This may be an important paradigm in the fetal rat lung because the activities of phosphatidylcholine-specific PLA2 and phosphatidylinositol-specific phospholipase C are higher in the reproductive tract of male fetal mice compared with female counterparts (34). Exogenously administered testosterone has also been shown to up-regulate the activity of these enzymes (36). Further, these findings were seen at d 18 gestation, a time when a surge in surfactant synthesis in this animal model begins (37). Together, these observations led us to hypothesize that enhanced DSPC hydrolysis by phospholipase activation may contribute to lower levels of surfactant in male fetuses. In support of this hypothesis, crude PLA2 activity, as determined by measuring [3H]-arachidonic acid release into medium, was observed to be consistently greater in male lung cells compared with females (Fig. 2A). These data suggest that there may be sex differences in the activities of the secretory or cytosolic forms of lung PLA2. Although there may be sex differences in these PLA2 subtypes, these observations did not translate into sex differences in DSPC degradation (Fig. 1A). Additional studies assessing [3H]-palmitic acid release and direct assays for exogenous calcium-independent PLA2 activity did not reveal significant gender-related differences. These latter studies were important because unlike the secretory or cytosolic forms of PLA2, calcium-independent PLA2 seems to be the primary PLA2 involved in regulating DSPC content in type II cells (38). Similarly, the activities of PC-PLC and PLD were not significantly different between male and females lung cells.

One factor that can affect both phosphatidylcholine turnover and biosynthesis is the availability of choline. Choline concentrations are 5-fold greater in the fetus than in the maternal circulation, and choline is actively taken up by fetal pretype II cells (39). Thus, choline availability is crucial for adequate surfactant synthesis. We observed that female mixed monolayer cultures had a greater capacity to transport choline compared with male cultures (Fig. 2A). It is difficult to explain the lack of differences shortly after cell isolation, although it may be that these cells require adequate time for equilibration and attachment to plastic surfaces before these differences can become apparent. Nevertheless, modest sex-specific differences were observed in choline uptake, which may partly explain differential rates of choline incorporation into DSPC reported in the current and past studies.

Prior radiolabeled studies also suggested sex differences in the biosynthetic pathway, but confirmation required examination of choline transport as described above, choline pool sizes, and enzymes involved in phosphatidylcholine synthesis. Two previous investigations failed to detect sex differences in CT activity in fetal lung (40, 41). It is notable, however, that in these studies enzyme analysis was conducted in a rabbit model by using tissue preparations from lung slices or whole lung, which may not reflect biosynthetic activity within the relevant cell populations. Consistent with these observations, we did not detect significant enzyme differences in whole lung (Table 4) or cultured fetal rat lung explants (data not shown). However, when cell populations are sufficiently enriched with fibroblasts and pretype II cells, sex-specific differences were uncovered (Tables 2 and 3). It is possible that these sex differences in the biosynthetic pathway could be due to greater proliferation of female fibroblasts compared with their male counterparts. To address this, we separately seeded male and female fibroblasts into cell culture and performed cell counts over a 72-h period and observed no substantial differences in growth rates between the sexes (data not shown). Interestingly, analysis performed in male and female purified pretype II cells revealed that sex-specific differences in CT activity were not identified (Table 5). These results led us to investigate whether soluble factors released from fibroblasts may modulate enzyme activity differently in male and female pretype II cells.

One factor released from fibroblasts that seems to play an integral role in stimulating surfactant phospholipid synthesis is FPF (12, 13). Corticosteroids increase FPF secretion in a sex-specific manner, whereas other regulators such as TGF-β and androgens inhibit its release (12, 13, 29, 34). We examined the role of these factors on CT function by incubating pretype II cells with cortisol-conditioned fibroblast medium with or without neutralizing TGF-β antibody. We added neutralizing antibody together with cortisol because corticosteroids have been shown to regulate TGF-β expression in lung fibroblasts (30, 31). Our results showing that cortisol-conditioned medium alone up-regulated enzyme activity in male and female pretype II cells suggest that FPF was active within the CDP-choline pathway, although only to a modest extent. Indeed, cortisol may also up-regulate biosynthesis at other control points within the pathway. In contrast, when pretype II cells were exposed to cortisol-conditioned fibroblast medium with neutralizing TGF-β antibody, male cells had significantly greater CT activity compared with females. These results support a sex-selective inhibitory role for TGF-β on enzyme activity under conditions of cortisol exposure. One possibility is that cortisol-induced TGF-β secretion was greater in male fibroblasts compared with females. Thus, in the presence of blocking antibody, this secretion is inhibited, resulting in higher levels of enzyme activity in male pretype II cells. However, unlike prior studies showing negative effects of the growth factor on DSPC synthesis, we did not observe a direct inhibitory effect of TGF-β on CT activity [data not shown (17)]. Therefore, TGF-β may act at other metabolic sites or act in an autocrine fashion within fibroblasts. An alternative explanation for our findings is that TGF-β may have suppressed cortisol-induced FPF release to a greater extent in male fibroblasts compared with females.

In addition to TGF-β, it is also possible that sex-specific differences in CT activity may be accentuated in vivo by androgens because these agents seem to down-regulate enzyme activity in adult lung (42). In addition to inhibiting FPF release, androgens decrease expression of the glucocorticoid receptor and enhance TGF-β binding to lung fibroblasts, thus providing additional mechanisms by which these agents antagonize surfactant synthesis (43, 44). Our preliminary studies show no inhibitory effects of dihydrotestosterone on CT activity within male and female pretype II cells after short-term (24 h) exposure (data not shown). However, these results do not necessarily exclude a suppressive role for these agents within the phosphatidylcholine biosynthetic pathway.

Collectively, the results to date suggest a more complex model underlying gender differences in surfactant phospholipid metabolism. The existing model suggests that certain soluble factors produced within lung fibroblasts are differentially expressed and released in male and female fetuses in response to sex hormones and growth factors. Thus, surfactant production within the male and female pretype II cell will be partly determined by the availability of these fibroblast factors, some of which may either activate or inhibit CT (45). The presence of circulating sex hormones may independently provide another level of regulation. Some of these sex hormones have recently been shown to regulate glucose transport, which also exhibits a sex dimorphism (46). Glucose, like choline, is an important precursor for surfactant synthesis. Thus, the emerging paradigm indicates that multiple metabolic control points involved in substrate utilization for phospholipid synthesis are differentially regulated between males and females and probably exist in vivo.