Dihydrotestosterone inhibits fetal rabbit pulmonary surfactant production.

Males have a higher morbidity and mortality for neonatal respiratory distress syndrome (RDS) than females, and respond less well to hormone therapy designed to prevent RDS by stimulating fetal pulmonary surfactant production. We have shown that male fetuses exhibit delayed production of pulmonary surfactant. We tested the hypothesis that the sex difference in fetal pulmonary surfactant production is under hormonal control. Pulmonary surfactant was measured as the saturated phosphatidylcholine/sphingomyelin ratio (SPC/S) in the lung lavage of fetal rabbits at 26 d gestation. There was an association between the sex of neighboring fetuses and the SPC/S ratio of the female fetuses, such that with one or two male neighbors, respectively, females had decreasing SPC/S ratios (P < 0.05). We injected dihydrotestosterone (DHT) into pregnant does from day 12 through day 26 of gestation in doses of 0.1, 1.0, 10, and 25 mg/d, and measured the SPC/S ratio in fetal lung lavage on day 26. In groups with the normal sex difference in fetal serum androgen levels (controls, 0.1 mg DHT/d) the normal sex difference in the SPC/S ratio was also present (females > males, P = 0.03). In the 1-mg/d group there was no sex difference in androgen levels and the sex difference in the SPC/S ratio was also eliminated as the female values were lowered to the male level. Higher doses of DHT (10, 25 mg/d) further reduced the SPC/S ratios. We injected the anti-androgen Flutamide (25 mg/d) from day 12 through day 26 of gestation. This treatment eliminated the normal sex difference in the lung lavage SPC/S ratio by increasing the male ratios to that of the females. We conclude that androgens inhibit fetal pulmonary surfactant production. An understanding of the mechanism of the sex difference in surfactant production may allow development of therapy that is as effective in males as in females for preventing RDS.

Organotypic differentiation of trypsin-dissociated fetal rat intestine.

These studies examined the potential for reorganization and differentiation of dissociated 18-day fetal rat intestine. Cultures of trypsin-dissociated fetal intestine were maintained in vitro for 1 week on a three-dimensional matrix, then transplanted into syngeneic hosts. When harvested after 4 weeks, these transplants consistently demonstrated organotypic differentiation. Spherical structures containing crypts with frequent mitotic figures and villi lined with columnar epithelium had formed. PAS staining demonstrated positive epithelial cell brush borders, goblet cells, and luminal contents. Significant levels of the microvillus membrane enzymes lactase, sucrase, maltase, and alkaline phosphatase were present in the luminal contents. Sucrase-isomaltase, an enzyme characteristic of postweaning small intestine, was demonstrated by immunoprecipitation and SDS-PAGE. Thus, both morphological and biochemical maturation occurred in the transplants.

Glucocorticoid regulation of DNA, protein and surfactant phospholipid in developing lung. Temporal relationship between growth and differentiation.

In contrast to the physiologic effects of glucocorticoids on lung saturated phosphatidylcholine (SPC) production, glucocorticoids have also been shown to inhibit fetal lung growth. We have found that at the time of increasing SPC production by the fetal rabbit, rat and chick lung there is a spontaneous fall in the DNA/protein ratio of the lung tissue, suggesting decreased cell proliferation similar to that which is seen with exogenous glucocorticoid treatment. Treatment of chick embryos with glucocorticoid causes a fall in the DNA/protein ratio and a rise in the SPC content of the lung; the antiglucocorticoid 11-deoxycortisol blocks both the spontaneous fall in the DNA/protein ratio and the rise in the lung SPC content. We conclude that there is a natural slowing of lung growth in response to endogenous glucocorticoid.

Cell culture of embryonic chick duodenal cells: preparation of epithelial-fibroblast bilayers and homotypic cultures of fibroblasts and epithelial cells.

A method for the primary cell culture of trypsin-dissociated embryonic chick duodenum is described. Both heterotypic (epithelial cells and fibroblasts together) and homotypic (highly enriched cultures of epithelial cells or fibroblasts alone) cell cultures were established. Dispersed duodenal epithelial cells and fibroblasts grown in 10% fetal bovine serum (FBS) spontaneously aggregated and proliferated as a bilayer of cells with the epithelial cells growing on top of the fibroblasts. Changing the serum supplement to 6% chicken serum (CS) and 4% FBS when the fibroblast monolayer reached confluence resulted in epithelial cell proliferation. Homotypic cultures of epithelial cells and fibroblasts were prepared and analyzed by scanning electron microscopy and transmission electron microscopy. Fibroblasts, isolated by differential adhesion and grown in 10% FBS, did not demonstrate measurable alkaline phosphatase activity. Homotypic epithelial cell cultures, isolated by floating them off the fibroblasts with collagenase, and maintained on collagen in 6% CS/4% FBS, demonstrated higher alkaline phosphatase-specific activity (16.1 +/- 2.3 U/mg protein) compared with epithelial-fibroblast bilayer cell cultures (12.1 +/- 1.3 U/mg protein).