Identification of Sex-Specific Differences in Surfactant Synthesis in Rat Lung

Abstract Delayed lung maturation and lower levels of surfactant phosphatidylcholine have been previously identified in male fetuses compared with female fetuses in several species. We investigated the mechanisms for sex differences in surfactant content by examining parameters of phosphatidylcholine turnover and biosynthesis; the latter was evaluated by measuring metabolic steps within the biosynthetic pathway. Compared with male lung cells, freshly isolated lung cells from female fetuses contained higher levels of disaturated phosphatidylcholine, a marker of surfactant lipid. Female mixed monolayer cultures exhibited a 71% increase in choline incorporation into disaturated phosphatidylcholine compared with male cultures. Male cultures exhibited significantly greater release of [3H]-arachidonic acid into the medium compared with females, suggesting sex differences in phospholipase activity. However, pulse-chase studies showed no sex differences in degradation of disaturated phosphatidylcholine, which was confirmed by assays of phospholipase A2, phosphatidylcholine-specific phospholipase C, and phospholipase D. Female mixed lung cells, however, had greater rates of cellular choline transport and activity of cytidylyltransferase, the rate-regulatory enzyme for phosphatidylcholine synthesis. Separate studies showed that exposure of sex-specific pretype II cell cultures to cortisol-stimulated fibroblast-conditioned medium plus transforming growth factor-β–neutralizing antibody stimulated cytidylyltransferase activity to a greater extent in male cells compared with female cells. These studies indicate that sex differences in surfactant phospholipid content are not due to differences in phospholipid turnover, but rather differential regulation of specific metabolic steps within the surfactant biosynthetic pathway. The data also support a role for transforming growth factor-β as a negative regulator of a key surfactant biosynthetic enzyme within male lungs. 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. (...truncated)