Yolk granules are differentially acidified during embryo development in the stick insect Carausius morosus

Abstract

Newly laid eggs of stick insects comprise a unique fluid ooplasm that is gradually partitioned into a number of yolk granules by invasion of secondary vitellophages. This study aimed at establishing how yolk granules become acidified in the course of embryonic development. Data show that acidified yolk granules are rather scarce and randomly distributed in vitellophages of early embryos, while they tend to increase gradually in number as development proceeds to completion. Yolk granule acidification is progressively more inhibited in the presence of increasing concentrations of chloroquine, monensin and bafilomycin. A pro-protease was identified cytochemically and by immunoblotting in yolk extracts of progressively more advanced embryos. A specific monoclonal antibody raised against this pro-protease helped to demonstrate that it is gradually processed to yield a lower molecular weight polypeptide as development proceeds to completion. This latter polypeptide was identified as a protease using electrophoresis in polyacrylamide gels containing yolk extracts. Simultaneous administration of a fluorescent substrate for cysteine protease and an acidotropic probe produced superimposable labelling patterns, suggesting that only acidified yolk granules possess a proteolytic activity. On the other hand, yolk granules probed simultaneously for acidification and latent pro-protease yielded labelling patterns partially superimposed. Pro-protease labelling is gradually lost as yolk granules are progressively more acidified during development. Distinct labelling patterns were also obtained in vitellophages processed for the simultaneous detection of pro-protease and protease, suggesting that the two activities are expressed by different yolk granule populations, and that one is gradually converted into the other as time goes by.

Calcium-regulated fusion of yolk granules is important for yolk degradation during early embryogenesis of Rhodnius prolixusStahl

“…If one of these three components is not found in a common vesicle, yolk degradation does not proceed. As highlighted in this paper, the YG population is not homogeneous and it has been extensively shown that YGs can vary in size, density, macromolecule content, and that not all of them undergo acidification during embryogenesis (Postlethwait and Giorgi, 1985;Wallace, 1985;Fagotto, 1995;Chestkov et al, 1998;McNeil et al, 2000;Fausto et al, 2001).…”

“…In the silkmoth Bombyx mori, the acid enzymes are only present in the small YGs (SYGs), whereas the yolk proteins are localized in the large ones (Yamahama et al, 2003). In the stick insect Carausius morosus, small YGs are frequently more acidic than large YGs and are often seen surrounding the large ones (Fausto et al, 2001). For the hard tick Boophilus microplus, proteolytic activity has also been correlated with differential acidification of YGs and the presence of small vesicles at the periphery of the egg (Abreu et al, 2004).…”

V-ATPase expression during development ofArtemia franciscanaembryos: potential role for proton gradients in anoxia signaling

“…Criel, 1991), formation of yolk platelets (Giorgi et al, 1999;Warner et al, 2002) and yolk degradation (Komazaki and Hiruma, 1999;Perona et al, 1988;Vallejo, 1985, 1989). Indeed, membrane-bound yolk platelets themselves should be considered, as they are acidified to varying degrees for both maintenance and degradation in other species (Abreu et al, 2004;Fagotto, 1995;Fausto et al, 2001) and contain very little P i in A. franciscana (Warner and Huang, 1979). An interesting commonality among all of these compartments is that their final luminal pH is set in part by the V-ATPase proton pump (Futai et al, 1998;Grabe and Oster, 2001;Nishi and Forgac, 2002), and thus this enzyme became the focal point of our research.…”

Serosa membrane plays a key role in transferring vitellin polypeptides to the perivitelline fluid in insect embryos

“…With the onset of embryonic development, this pro-enzyme is gradually converted to an active proteolytic enzyme by progressive acidification of the yolk granules (Fagotto 1990;Nordin et al 1991;Fagotto & Maxfield 1994;Fausto et al 2001). Following activation of the pro-protease, vitellin polypeptides are degraded by limited proteolysis to generate a number of lower molecular weight Vt cleavage products.…”