Transient activation of calcineurin is essential to initiate embryonic development in Xenopus laevis

Abstract

At fertilization, an increase of cytosolic calcium ions (Ca2+) triggers various activation responses in animal eggs. In vertebrates, these responses include exit from metaphase arrest in meiosis II (MII exit) and cortical remodelling initiated by cortical granule exocytosis. Although the essential requirement of Ca2+/calmodulin-dependent protein kinase II for inducing MII exit has been documented, a role of the Ca2+/calmodulin-dependent protein phosphatase calcineurin in egg activation has not been investigated. Here we show, using cell-free extracts from unfertilized eggs of Xenopus laevis, that calcineurin is transiently activated immediately after Ca2+ addition to a concentration that induces MII exit. When calcineurin activation is inhibited, cyclin-dependent kinase 1 (Cdk1) inactivation by means of cyclin B degradation is prevented and sperm chromatin incubated in the extracts remains condensed. Similarly, if calcineurin is inhibited in intact eggs, MII exit on egg activation is prevented. In addition, the activation contraction in the cortex is suppressed whereas cortical granule exocytosis occurs. We further demonstrate that, when a high level of calcineurin activity is maintained after activation, growth of sperm asters is prevented in egg extracts and, consistently, migration of male and female pronuclei towards each other is hindered in fertilized eggs. Thus, both activation and the subsequent inactivation of calcineurin in fertilized eggs are crucial for the commencement of vertebrate embryonic development.

Transitioning from egg to embryo: Triggers and mechanisms of egg activation

“…The Ca 2ϩ -and calmodulin-dependent phosphatase Calcineurin (CaN) was shown to be involved in egg activation of Drosophila (Horner et al, 2006;Takeo et al, 2006; and see below). CaN has recently been shown to play a role in Xenopus egg activation as well (Mochida and Hunt, 2007;Nishiyama et al, 2007b). Upon egg activation in Xenopus, CaN is rapidly and transiently activated, independently of CaMKII (Mochida and Hunt, 2007;Nishiyama et al, 2007b).…”

“…CaN has recently been shown to play a role in Xenopus egg activation as well (Mochida and Hunt, 2007;Nishiyama et al, 2007b). Upon egg activation in Xenopus, CaN is rapidly and transiently activated, independently of CaMKII (Mochida and Hunt, 2007;Nishiyama et al, 2007b). Once activated, CaN reverses global mitotic phosphorylations (Mochida and Hunt, 2007).…”

“…Once activated, CaN reverses global mitotic phosphorylations (Mochida and Hunt, 2007). Furthermore, transient activity of CaN is necessary for the inactivation of MPF and exit from meiosis II in Xenopus (Nishiyama et al, 2007b). While the precise targets of CaN remain unknown, in vitro CaN directly dephosphorylates Cdc20 and a component of the APC, Apc3 (Mochida and Hunt, 2007).…”

“…Specifically, protamines are removed and maternal histones are added to the paternal chromatin (Horner et al, 2006;Bonnefoy et al, 2007) but DNA replication, full chromatin decondensation, and the formation of the sperm aster do not occur (Horner et al, 2006). In Xenopus, constitutively active Calcineurin prevents sperm aster formation (Nishiyama et al, 2007b). This finding suggests that acquisition of competence by the egg's cytoplasm for reorganizing the sperm nucleus into a male pronucleus is a feature of egg activation, and also that it is downstream of the calcium signal.…”

“…In Xenopus, a connection between Ca 2ϩ and cytoskeletal changes that occur upon egg activation has been forged through CaN: rearrangement of the cytoskeleton in the cortical areas of the egg does not occur when CaN is inhibited in Xenopus (Nishiyama et al, 2007b). Therefore, it is possible that the ability to stimulate or enhance APC activity (through CaN in Xenopus or Cortex in Drosophila) is necessary for proper remodeling of the cytoskeleton upon egg activation.…”