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The pulmonary surfactant system matures upon pipping in the freshwater turtle Chelydra serpentina

Sonya D. Johnston^1,2, Christopher B. Daniels^1,2, David Cenzato², Jeffrey A. Whitsett³ and Sandra Orgeig^1,2,*

¹ Department of Physiology and
² Department of Environmental Biology,University of Adelaide, Adelaide SA 5005, Australia,
³ Division of Pulmonary Biology and Neonatology, Children’s Hospital Medical Center, Cincinnati OH 45229–3039, USA

View larger version (175K): [in a new window]   Fig. 1. Immunohistochemical staining of TTF-1 in the developing lung of the snapping turtle. (A–C) TTF-1 was detected in the nuclei of epithelial cells of the trabeculae (T) and gas-exchange area (arrows). The greatest degree of staining occurring at day 61 of incubation (A), and was seen in fewer cells after pipping (B), with the lowest number and intensity after hatching (C). (D–F) Tissues were incubated without primary antibody. Immunostaining was not observed at day 61 (D), after pipping (E) or after hatching (F). Scale bars, 50 µm.

View larger version (66K): [in a new window]   Fig. 2. High-power photomicrograph demonstrating immunohistochemical staining of TTF-1 in the developing lung of the snapping turtle. TTF-1 staining was confined to nuclei of cells within the gas-exchange region and trabecular epithelium in snapping turtles at day 61 of incubation (A), after pipping (B) and after hatching (C). Regions depicted here correspond to areas demarked by the arrows in the corresponding panels of Fig. 1. Scale bars, 20 µm.

View larger version (182K): [in a new window]   Fig. 3. Immunohistochemical staining of mature SP-B in the developing lung of the snapping turtle. (A–C) SP-B was detected in the cytoplasm of subsets of epithelial cells in the gas-exchange area (arrows) at day 61 of incubation (A), after pipping (B) and after hatching, when staining intensified (C). (D–F) Tissues were incubated without primary antibody. Immunostaining was not observed at day 61 (D), after pipping (E) or after hatching (F). T, trabeculae. Scale bars, 50 µm.

View larger version (80K): [in a new window]   Fig. 4. High-power photomicrograph demonstrating immunohistochemical staining of SP-B in the developing lung of the snapping turtle. SP-B staining was detected in the cytoplasm of subsets of epithelial cells within the gas-exchange region of snapping turtles at day 61 of incubation (A), after pipping (B) and after hatching (C). Regions depicted here correspond to areas demarked by the arrows in the corresponding panels of Fig. 3. Scale bars, 20 µm.

View larger version (28K): [in a new window]   Fig. 5. The absolute amounts of total (A) and disaturated (B) phospholipids and cholesterol (C) in pulmonary surfactant from the developing lung of the snapping turtle. Values are per mg of dry lung (means ± S.E.M.). Paired symbols indicate a significant difference between adjacent groups. (A) *P=7.6x10–8, P=0.05; (B) *P=1.5x10–5; (C) *P=0.033.

View larger version (27K): [in a new window]   Fig. 6. The relationship between phospholipids and cholesterol in pulmonary surfactant from the developing lung of the snapping turtle, demonstrating (A) disaturated phospholipids (DSP) expressed as a percentage of total phospholipids (PL), (B) the cholesterol (Chol)/PL ratio and (C) the Chol/DSP ratio. Values are means ± S.E.M. Paired symbols indicate a significant difference between adjacent groups. (A) *P=0.019, (B) *P=2.6x10–4, (C) *P=3.2x10–4.