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The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.

The term “neurosteroid” refers to both classic and unique steroid molecules that are synthesized from cholesterol (C) by the central and peripheral nervous systems of higher vertebrates. Therein, they accumulate and modulate nervous activity by a variety of mechanisms other than the classic steroid receptor-mediated modulation of genomic activity, although such may also be involved. Since the insect nervous system expresses ecdysteroid receptors and responds both directly and developmentally to ecdysteroids, the possibility of ecdysteroidogenesis in the pupal and adult central and peripheral nervous system of Manduca sexta and the nervous system of Drosophila melanogaster larvae was investigated. The endogenous concentrations of the critical, dietary-derived Δ 5,7 -sterols ergosterol and 7-dehydrocholesterol (7dC) remained 10 to 20-fold higher in the Manduca pupal and adult nervous tissues than was found in the larval hemolymph at the cessation of feeding. In addition, it was determined that the Manduca pupal nervous system, but not that of the adult, could synthesize 3 H/ 14 C-7dC or 3 H-7-dehydro-25-hydroxycholesterol ( 3 H-7d25C) from 3 H/ 14 C-cholesterol ( 3 H/ 14 C-C) or the polar sterol substrate 3 H-25-hydroxycholesterol ( 3 H-25C), respectively. However, none of the nervous system samples from the two species and the several stages analyzed, a small window of neural development in these insects, were capable of incorporating any of the above tracer precursor sterols into a radiolabelled ecdysteroid, i.e. less than 0.0005%. Thus, the absence of neurosteroidogenesis by the insect nervous system stands in sharp contrast to previously described nervous system steroid hormone biosynthesis by the mammalian nervous system.

During normal pupal-adult development, programmed cell death of the prothoracic gland of Manduca sexta proceeds via apoptosis. By employing the DNA laddering technique, the earliest sign of DNA fragmentation in the cells comprising the gland occurred on Day 5 of pupal-adult development and DNA fragmentation peaked 1 day later. Since juvenile hormone (JH) is known to prevent adult development and since prothoracic gland degeneration occurs during the initial stages of adult development, we wished to determine the possible role of JH in prothoracic gland maintenance. JH was injected into pupae and the glands analyzed by DNA laddering and in situ TUNEL labeling. The administration of JH prevented apoptosis of the prothoracic gland even 11 days after JH injection into young pupae. The prothoracic glands remained intact and their ability to synthesize ecdysteroids was maintained at a fairly active level as ascertained by radioimmunoassay after in vitro incubation. The control glands had degenerated by this time and were almost devoid of ecdysteroidogenesis capability. Ultrastructural analysis confirmed that JH-treated prothoracic gland cells were rescued from the apoptotic sequence, i.e., nuclear condensation, cytoplasmic budding, and cell fragmentation. They exhibited a preserved smooth endoplasmic reticulum and intercellular channel system typical of active prothoracic gland cells. The composite data suggest that JH can both maintain and stimulate the prothoracic gland. Therefore, during normal pupal-adult metamorphosis the absence of JH is prerequisite for both the initiation and completion of prothoracic gland degeneration.

Studies in vitro revealed that intact ring glands of Drosophila melanogaster convert tritiated cholesterol (C) and 25-hydroxycholesterol (25C) via 7-dehydrocholesterol (7dC) and 7-dehydro-25-hydroxycholesterol (7d25C), respectively, to ecdysone (E) and 2-deoxyecdysone (2dE), while both intact and homogenized ovaries synthesize only 2dE from these precursors. Emulsified 7d25C was incorporated directly into ecdysteroids by these tissue preparations at a much greater rate than was 7d25C made in situ from 25C. To probe the basis of the biochemical defect in the ecdysteroid deficient conditional mutant ecdysoneless (ecd1), the differential incorporation into ecdysteroids of C (via 7dC), and particularly of 25C (via 7d25C), was measured relative to that observed after the incubation of 7d25C directly with both wild type and mutant tissues in vitro at 30 degrees C, the restrictive temperature. Both C and 25C were equally 7,8-dehydrogenated in situ to 7dC or 7d25C, respectively, by both wild type and mutant tissues at 30 degrees C. However, the rate of subsequent conversion of either of these delta 5,7-sterol intermediates synthesized in situ to ecdysteroids was reduced an average of 50% in the mutant tissues relative to the wild type. Yet, when emulsified 7d25C was incubated directly with either the wild type or mutant tissues at the restrictive temperature, the amplified rate of conversion of the freely available 7d25C to ecdysteroid by these tissues was identical. These data suggest that the defect in ecd1 tissue-mediated ecdysteroidogenesis does not involve a "hit" on any of the enzymes involved in either the 7,8-dehydrogenation of C or 25C or in the subsequent oxidation of 7d25C or 7dC to ecdysteroid. Rather, the mutation appears to affect the expression of a gene governing the translocation of delta 5,7-sterol intermediates from the subcellular compartment where they are synthesized and/or stored to the site of subsequent oxidation to ecdysteroid.

The insect brain neuropeptide, prothoracicotropic hormone (PTTH), elicits the molting process by stimulating ecdysteroidogenesis in the prothoracic glands. Changes in the subcellular distribution of PTTH during the fifth larval instar and larval-pupal metamorphosis of Bombyx were investigated using immunogold electron microscopy and computerized image analysis. A time-resolved fluoroimmunoassay was employed to quantify the hemolymph PTTH titer and the hemolymph ecdysteroid titer was determined by radioimmunoassay. Immunoreactivity was apparent only in the cytoplasm of two pairs of dorsal-lateral neurosecretory cells (La) in the brain, the prothoracicotropes, and their axons that traverse the peripheral area of the brain neuropil and then enter the neurohemal lobes of the corpora cardiaca and end in the corpora allata. Immunospecificity was evident as noted by the restriction of the 5-nm gold particles to the neurosecretory granules. Synaptic contacts were noted frequently between the dendritic collaterals of the prothoracicotropes and neighboring collaterals from other neurons. The morphological data suggest that PTTH is synthesized and released throughout the fifth larval instar. The concentration of gold particles in the neurosecretory granules and the number of neurosecretory granules increased five times during the fifth instar, and these increases were correlated with surges in the hemolymph PTTH and ecdysteroid titers. The data suggest that PTTH biosynthesis, secretory granule formation, and secretion are three continuous and almost synchronized events.

The ultrastructure of the corpus allatum of theapterous mutantsap 4 andap 56f ofDrosophila melanogaster during larval-pupal-adult metamorphosis and adult life was correlated with the gland's ability to synthesize juvenile hormone in vitro. During the early wandering period of the third instar of both mutants, a high concentration of smooth endoplasmic reticulum, mitochondria and mitochondrion-scalariform junction complexes are typical features of an active corpus allatum cell. Juvenile hormone biosynthesis by the glands is high at that time and, in fact, only slightly lower than that of wild type glands. In contrast to the wild type gland, the cells of the pupal and pharate adult corpus allatum of both mutants contains highly electron dense mitochondria with tubular cristae but no whorls of smooth endoplasmic reticulum nor glycogen clusters. The frequency and size of the lipid droplets, putatives depots of the juvenile hormone precursors, in cells of theap 56f gland is a function of the insect's age, but both are lower than in wild type gland cells. Juvenile hormone biosynthesis by both mutant glands remains at the basal level when compared to increased synthesis by the wild type gland. The frequency and density of lipid droplets in cells of theap 4 corpus allatum are much lower than in theap 56f glands. During adult life, the ultrastructural profile of theap 56f corpus allatum is similar to that of the wild type gland although the in vitro production of juvenile hormone by the former is much lower than that of the wild type gland. The ultrastructural features of the adult corpus allatum ofap 4 homozygotes reveal precocious degeneration and support the view that this non-vitellogenic mutant is a juvenile hormone deficient mutation.

In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29 degrees C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18 degrees C to 29 degrees C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29 degrees C following dissection from wandering third instar larvae reared at 18 degrees C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.

Insect molting and metamorphosis are elicited by a class of polyhydroxylated steroids, ecdysteroids, that originate in the prothoracic glands (PGs). Prothoracicotropic hormone stimulation of steroidogenesis by the PGs at the cellular level involves both calcium and cAMP. Cell-to-cell communication mediated by gap junctions may play a key role in regulating signal transduction by controlling the transmission of small molecules and ions between adjacent cells. This is the first report of gap junctions in the PGs, the evidence obtained by means of SEM, thin sections and freeze-fracture replicas.