Ultrastructural evidence of a vesicle-mediated mode of cell degranulation in chicken chromaffin cells during the late phase of embryonic development

Abstract

In the present investigation, we attempted to determine whether ultrastructural features indicative of a vesicle-mediated mode of cell secretion were detectable in chick chromaffin cells during embryo development. The adrenal anlagen of domestic fowls were examined at embryonic days (E) 12, 15, 19 and 21 by electron microscopy quantitative analysis. Morphometric evaluation revealed a series of granule and cytoplasmic changes highly specific for piecemeal degranulation (PMD), a secretory process based on vesicular transport of cargoes from within granules for extracellular release. At E19 and E21 we found a significant peak in the percentage of granules exhibiting changes indicative of progressive release of secretory materials, i.e. granules with lucent areas in their cores, reduced electron density, disassembled matrices, residual cores and membrane empty containers. A dramatic raise in the density of 30-80-nm-diameter, membrane-bound, electron-dense and electron-lucent vesicles--which were located either next to granules or close to the plasma membrane--was recognizable at E19, that is, during the prehatching phase. The cytoplasmic burst of dense and clear vesicles was paralleled by the appearance of chromaffin granules showing outpouches or protrusions of their profiles ('budding features'). These ultrastructural data are indicative of an augmented vesicle-mediated transport of chromaffin granule products for extracellular release in chick embryo chromaffin cells during the prehatching stage. In conclusion, this study provides new data on the fine structure of chromaffin cell organelles during organ development and suggests that PMD may be part of an adrenomedullary secretory response that occurs towards the end of chicken embryogenesis. From an evolutionary point of view, this study lends support to the concept that PMD is a secretory mechanism highly conserved throughout vertebrate classes.

Fig. 1

Electron micrographs of chicken chromaffin cells at different stages of embryonic development. (a) In chromaffin cells viewed at E12, resting unaltered granules are prevailing. This cell presents a predominance of homogeneous, highly osmiophilic granules. Some finely particulate, moderately electron-dense granules are also identifiable (asterisks). (b) At E19, the number of type 2 granules (2) greatly increases, paralleled by a diminution of type 1 (1) granules. Changes in the granule repertoire are accompanied by an outburst of electron-dense (arrows) and electron-lucent (arrowheads) vesicles located close to the granules, free in the cytosol or near the plasma membrane. (c) This chromaffin cell viewed at E21 shows a large type 2 granule (asterisk) with residual core and partially disassembled matrix. (d) Numerous vesicles containing electron-dense material (arrows) can be observed in the cytoplasm of this cell examined at E19. Bar = 0.5 µm (a); 0.2 µm (b); 0.3 µm (c); 0.1 µm (d).

Fig. 2

Graph illustrating the percentage of chromaffin granules exhibiting degrees of piecemeal degranulation in the chromaffin cells of chicken embryos at different incubation days (12, 15, 19, 21). ‘Type 1 granules’ are resting, unaltered granules; ‘type 2 granules’ are granules with changes indicative of progressive release of secretory materials. Each column represents mean ± SD. Asterisks and empty circles indicate values significantly different from the respective values at E12 and E15 (P < 0.01).

Fig. 3

Graph illustrating the areas of type 1 and type 2 chromaffin granules in the chromaffin cells of chicken embryos at different incubation days (12, 15, 19, 21). Each column represents mean ± SD. Asterisks indicate values significantly different from the respective values at E12 and E15 (P < 0.01).

Fig. 4

Graph highlighting the differences in the density of 30–80-nm cytoplasmic vesicles, calculated as number of vesicles per µm² of net cytoplasmic area, in chromaffin cells of chicken embryos at different incubation days (12, 15, 19, 21). Each column represents mean ± SD. The asterisk indicates value significantly different from E12 and E15 (P < 0.01).

Fig. 5

Graph illustrating the topographic distribution of 30–80-nm vesicles in the chromaffin cells of chicken embryos at different incubation days (12, 15, 19, 21). Vesicle densities are calculated as in Fig. 4. Bars refer to vesicle density within a distance of 100 nm from (i) type 1 granules, (ii) type 2 granules, (iii) the plasma membrane, (iv) the perinuclear membrane. Each column represents mean ± SD.

Fig. 6

High magnification electron micrographs illustrating the ultrastructural details of chromaffin granules in chicken chromaffin cells at E19 (a,b). Two granules present budding protrusions, consisting in evaginations of the perigranule membranes (arrows) that appear filled by the same electron-dense material stored inside the granules. = 0.1 µm (a,b).

Fig. 7

Graph highlighting the differences in the percentage of granules exhibiting ‘budding features’ in chromaffin cells of chicken embryos at different incubation days (12, 15, 19, 21). Each column represents mean ± SD. The asterisk indicates value significantly different from E12 and E15 (P < 0.01).