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During the 9-day pupal period of Tenebrio molitor (the mealworm beetle), the cells of the male accessory glands undergo divisions for 7 days. There are two maxima in the mitotic activity in the glands in vivo, one at 1 day and the other at 4 days. The latter peak coincides with the large surge of ecdysterone occurring in the pupal stage. By the use of in vitro culture techniques, it has been demonstrated that the first bout of mitosis in both glands proceeds in basal medium, while the second bout of mitosis requires a physiological level of ecdysterone. Ecdysone was less effective than ecdysterone. Sensitivity to ecdysterone did not change significantly between Day 1 and Day 4 of the pupal stage. The results are discussed in relation to the effects of ecdysterone on cell division in mesodermal and ectodermal derivatives.

The cells of the bean-shaped accessory glands of mealworms proliferate through the first 7 days of the 9-day pupal stage. Immediately after larval-pupal ecdysis, 25-27% of the cells were in the G1 phase, 60-65% were in the G2 phase, and the balance were in S phase. Over the first 4 days of normal development, the S fraction gradually increased, to reach its highest level in the mid-pupa at the time of the major ecdysteroid peak (Delbecque et al., 1978). Thereafter, the S fraction declined until over 95% of the cells had accumulated in G2 on Day 8. When 0-day pupal glands were explanted into Landureau's S-20 medium for 6 days, the G1 fraction remained fairly constant (25-30%) while S and the G2 fractions fluctuated. On the first day in vitro, the G2 fraction declined and the S fraction rose. On the second day in basal media, the S fraction fell and G2 rose correspondingly until 70% of the cells reached G2 when cycling stopped on the third day. With addition of 20-hydroxyecdysone to 0-day cultures, the S fraction increased quite sharply. It remained large for all 6 days of the experiment in the continuing presence of hormone. A 1-day pulse of hormone produced a transient increase in S. We blocked cell cycling with hydroxyurea in a stathmokinetic experiment and showed that 20-hydroxyecdysone accelerated the flow of cells from the G2 phase to the G1 phase by 2.5-fold. An increase in the G1 fraction was detected within 10 hr of hormone administration and the effect was dose-dependent with an ED50 of 5 X 10(-7) M for 20-hydroxyecdysone. We conclude that 20-hydroxyecdysone acts at a control point in the G2 phase. Incubation of the glands with 20-hydroxyecdysone for only 30-60 min followed by washout stimulated the flow from G2 to G1 and the effect persisted after transfer of the tissues to hormone-free media. Dose-dependent stimulation also occurred with ponasterone A (ED50 3 X 10(-9] but not with cholesterol.

During the pupal stage of Tenebrio molitor, the accessory reproductive glands of males grow by cell division. Within the secretory epithelium of the bean-shaped accessory glands (BAGs), cell numbers triple. In the tubular accessory glands (TAGs), the increase is 14-fold. There are two mitotic maxima in each gland. The first maximum occurs at 1-2 days while the second is at 4-5 days. The second maximum coincides with the major ecdysteroid peak described by Delbecque et al. [Dev. Biol. 64, 11-30 (1978)]. Nuclei were isolated from TAGs during the pupal mitotic bouts and during mitotic inactivity in the adult. After Feulgen or propidium iodide staining, the DNA content of these nuclear populations was measured by absorption cytophotometry or by fluorescence flow cytometry, respectively. The proportion of cells in each phase of the cycle was calculated using an iterative model. After mitoses have ended in the late pupa, the cells were arrested in G2. [3H]Thymidine was injected into 1- and 4-day pupae to pulse-label cells of the TAGs. After allowing various periods from 4 to 60 hr for cells to progress through G2 to reach mitosis, fractions of labelled mitoses were determined by autoradiography. From the combined cytometric and autoradiographic data, the duration of each phase of the cell cycle was calculated assuming the population was in exponential growth. Cell cycles in 4-day pupal TAGs take 48 hr. G1, S, G2, and, M lasted 13, 14, 17, and 4 hr, respectively.