Capacity of insect (Manduca sexta) prothoracic glands to secrete ecdysteroids: relation to glandular growth

The capacity of prothoracic glands to secrete ecdysteroids changes during the last larval stadium of the tobacco hornworm, Manduca sexta. In the present study, the protein content of prothoracic glands was observed to change significantly during this time period. Peaks in glandular protein content occurred on Day 4 (15.4 micrograms/gland) and Day 7 (14.6 micrograms/gland). These correspond to times of maximal ecdysteroid secretion in vitro. Ecdysteroid secretion in vitro was determined as a function of glandular protein content for Day 1, 3, and 7 glands. For unstimulated glands, secretion increased from 0.05 ng/micrograms on Day 1, to 0.54 ng/micrograms on Day 3, to 1.37 ng/micrograms on Day 7. For glands incubated with big PTTH, secretion increased from 0.27 ng/micrograms on Day 1, to 2.05 ng/micrograms on Day 3, to 2.60 ng/micrograms on Day 7. The results suggested that developmental changes in secretory capacity are influenced by both the amount and type of glandular proteins. Glandular protein metabolism was assessed by monitoring the incorporation of [35S]methionine. A time course study revealed the rate of incorporation for Day 3 and Day 5 glands was significantly greater than the rate for Day 1 and Day 7 glands. Electrophoretic separation of radiolabeled glandular proteins revealed developmental changes in the pattern of protein synthesis. However, a band whose intensity changed in parallel with developmental changes in glandular secretory capacity was not detected. Finally, incorporation of BrdU by cells of the prothoracic glands was assessed using immunohistochemistry: Incorporation of BrdU was not observed on Days 1 or 7, occurred in only a few cells on Day 5, and was most pronounced on Day 3 (12.3% of the cells were labeled). The combined results indicate that changes in ecdysteroidogenic capacity are associated with (a) a change in glandular protein content, (b) a change in the types of proteins synthesized by prothoracic glands, and (c) a temporally restricted pulse of DNA synthesis, the latter being a possible indicant of cell proliferation.