The prothoracic glands ofManduca sexta:a microscopic analysis of gap junctions and intercellular bridges

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Abstract
The insect prothoracic gland is an ecdysteroidogenic (molting hormone) structure that under the control of a brain neuropeptide is responsible for the surges in ecdysteroid titer required for cellular reprogramming, molting and metamorphosis. The glands are stimulated by the brain neurohormone via a signal transduction cascade that involves cAMP, Ca2+/calmodulin and protein kinases. Since the precision of the ecdysteroid surge is vital for the coordination of those biochemical processes encompassing molting, an investigation was initiated to provide basic information for a future analysis of how the 200–250 cells of the M. sexta prothoracic gland communicate to assure a rapid, uniform and temporally correct surge of ecdysteroids. An analysis of gap junctions between cells of the prothoracic glands was undertaken using scanning (SEM) and transmission electron microscopy (TEM) as well as freeze fracture. The SEM data revealed that during the fifth larval instar the cell size, shape and surface morphology underwent dramatic changes, e.g., the cell size increased from 30–40 μm in diameter on day 1 to about 65 μm on day 7. In addition, there was the development of cell-to-cell major connecting areas and intercellular bridges. TEM analysis demonstrated that both the major connecting areas and intercellular bridges contain interdigitating cellular processes from neighboring cells. Typical gap junctions are distributed in an organized manner, occurring on limited portions of the cell membrane, with preferential localization close to the distal ends of these processes. They are septilaminar structures with a central cleft 2–3 nm wide and electron-dense strata that appear to be symmetrical. Numerous multilamellar bodies are often seen in these areas. The size and frequency of both gap junctions and multilamellar bodies appear to be correlated with the developmental stage and physiological state of the insect. Freeze fracture images of the region of contact between interdigitating processes reveals clusters of particles on the E face. These clusters represent gap junctions and are similar to gap junction clusters reported for other insect tissues. The clusters are often irregular in shape, and the particles are always randomly arranged. Many of the particles are elongated, suggestive of dimers or the bridging of adjacent particles, and these elongated particles are matched by a corresponding pattern of pits found on the P face. These gap junctions may promote the synchronization of secretory activity of the cell comprising the prothoracic glands.