Increase of intracellular Ca ²⁺ and relocation of E-cadherin during experimental decompaction of mouse embryos

Abstract

To determine the role of intracellular Ca 2؉ in compaction, the first morphogenetic event in embryogenesis, we analyzed preimplantation mouse embryos under several decompacting conditions, including depletion of extracellular Ca 2؉ , blocking of Ca 2؉ channels, and inhibition of microfilaments, calmodulin, and intracellular Ca 2؉ release. Those treatments induced decompaction of mouse morulae and simultaneously induced changes in cytosolic free Ca 2؉ concentration and deregionalization of E-cadherin and fodrin. When morulae were allowed to recompact, the location of both proteins recovered. In contrast, actin did not change its cortical location with compaction nor with decompaction-recompaction. Calmodulin localized in areas opposite to cell-cell contacts in eight-cell stage embryos before and after compaction. Inhibition of calmodulin with trifluoperazine induced its delocalization while morulae decompacted. A nonspecific rise of intracellular free Ca 2؉ provoked by ionomycin did not affect the compacted shape. Moreover, the same decompacting treatments when applied to uncompacted embryos did not produce any change in intracellular Ca 2؉ . Our results demonstrate that in preimplantation mouse embryos experimentally induced stage-specific changes of cell shape are accompanied by changes of intracellular free Ca 2؉ and redistribution of the cytoskeleton-related proteins E-cadherin, fodrin, and calmodulin. We conclude that intracellular Ca 2؉ specifically is involved in compaction and probably regulates the function and localization of cytoskeleton elements.Extracellular calcium is crucial during the preimplantation development of mammalian embryos because of its role in cell-cell adhesion (1-5). Intracellular free calcium ([Ca 2ϩ ] i ) plays a pivotal role in triggering early events related to fertilization, namely, oocyte maturation, cortical granule exocytosis, and egg activation (6-19). However, little is known about the role of [Ca 2ϩ ] i during the following steps of mammalian development. Indirect evidence for a regulatory role was provided in experiments where development of mouse embryos was delayed by treatments with calcium channel blockers or with an inhibitor of calmodulin (20). It was only recently that [Ca 2ϩ ] i changes were studied directly during early mouse development. An endogenous increase in Ca 2ϩ -releasing activity leading to Ca 2ϩ transients was observed during the first mitotic division (21). Moreover, the rate of cavitation and cell division was either accelerated or delayed by experimentally induced elevation or reduction of [Ca 2ϩ ] i , respectively (22-24). Stacheki and Armant (24) suggested that in mouse embryos, inositol 1,4,5-trisphosphate-sensitive and ryanodinesensitive Ca 2ϩ stores exist at the morula stage and that calmodulin is involved in mediating the Ca 2ϩ signaling effects to its distal targets.Despite these temporal effects, no other developmental events have been examined so far under conditions where [Ca 2ϩ ] i has been manipulated and therefore t...

Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

“…6B), suggest that anandamide primarily influences N-type Ca 2ϩ channels in the blastocyst trophectoderm. This is consistent with previous observations that Ca 2ϩ signaling is crucial for blastocyst formation (28) and that N-type Ca 2ϩ channels are functionally coupled to the stimulating effects of heparin-binding EGF-like growth factor (HB-EGF) in blastocyst development and outgrowth (22).…”

Blastomere biopsy for PGD delays embryo compaction and blastulation: a time-lapse microscopic analysis

“…During cleavage-stage biopsies, Ca +2 /Mg + 2 -free medium is used in order to loosen cell-to-cell adhesion and facilitate selected blastomere removal. Several murine studies showed that Ca +2 depletion caused remodeling of the cellular cytoskeleton, inevitably affecting compaction [35][36][37][38]. Furthermore, artificial breaching of the zona pellucida by means of the laser system to enable blastomere biopsy may increase local temperature for a few milliseconds and potentially impact subsequent development [39], although Taylor et al had shown that laser pulse does not influence embryo development when the size of the hole is controlled [40][41][42][43].…”

Maternal β-catenin and E-cadherin in mouse development

“…E-cadherin mediates compaction of the individual blastomeres in the 8-cell stage embryo, and this adhesion triggers the development of the trophectoderm and other epithelial cell layers at later developmental stages (Gumbiner, 1996). In embryos, mutation of E-cadherin, perturbation of the function of molecules interacting with E-cadherin, or disturbance of the compaction process, all lead to the relocation of E-cadherin, and of the molecules interacting with it (Clayton et al, 1999;Ohsugi et al, 1997;Pey et al, 1998).…”