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Biol Bull 141: 527-540. (December 1971)
© 1971 Marine Biological Laboratory
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IN VITRO DEVELOPMENT OF INSECT TISSUES. I. A MACROMOLECULAR FACTOR PREREQUISITE FOR SILKWORM SPERMATOGENESIS

MICHAEL P. KAMBYSELLIS 1 and CARROLL M. WILLIAMS 1

1 The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138

1. "Naked" germinal cysts removed from the testes of diapausing Cynthia or Cecropia pupae and cultured in vitro undergo meiosis and spermatogenesis only when the culture medium contains a "macromolecular factor" (MF) which is present in insect blood. The factor in question is undialyzable, heat-sensitive, and interchangeable among the three genera of saturniid silkworms which were studied. Its partial purification was achieved by ammonium sulfate fractionation and by precipitation at low ionic strengths.

2. In appropriate experiments, ecdysone was found to have no obvious effect on cultures of naked cysts and was neither able to replace nor enhance the activity of MF in the in vitro assay.

3. MF is present in the blood plasma of both male and female pupae; its titer undergoes large and systematic changes when diapausing Cynthia pupae are stored at 25° C for up to six months.

4. In plasma collected from uninjured pupae, MF was routinely present except during a brief period 8 to 10 weeks after pupation. However, even in that case, substantial MF activity appeared in the blood during the first two days after an integumentary injury.

5. Circumstantial evidence is presented that MF is synthesized and secreted by one or more types of hemocytes when the latter are activated as, for example, by integumentary injury. Presumably because of their ability to secrete MF and thereby to condition the medium, the hemocytes were the only class of cell which could be cultured in Grace's medium without the addition of any macromolecules.

6. MF-like activities were demonstrated in assays of heat-treated calf serum, fetal calf serum, and newborn calf serum. It is not yet known whether these activities are related to the "serum factors" prerequisite for the successful culture of mammalian cells.

7. For these several reasons it is conjectured that MF or MF-like materials have heretofore been present in virtually all of the media which have sustained the successful culture of insect cells and tissues.




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