Release from meiotic arrest in ascidian eggs requires the activity of two phosphatases but not CaMKII

Abstract

The fertilising sperm triggers a transient Ca 2+ increase that releases eggs from cell cycle arrest in the vast majority of animal eggs. In vertebrate eggs, Erp1, an APC/C cdc20 inhibitor, links release from metaphase II arrest with the Ca 2+ transient and its degradation is triggered by the Ca 2+ -induced activation of CaMKII. By contrast, many invertebrate groups have mature eggs that arrest at metaphase I, and these species do not possess the CaMKII target Erp1 in their genomes. As a consequence, it is unknown exactly how cell cycle arrest at metaphase I is achieved and how the fertilisation Ca 2+ transient overcomes the arrest in the vast majority of animal species. Using live-cell imaging with a novel cyclin reporter to study cell cycle arrest and its release in urochordate ascidians, the closest living invertebrate group to the vertebrates, we have identified a new signalling pathway for cell cycle resumption in which CaMKII plays no part. Instead, we find that the Ca 2+ -activated phosphatase calcineurin (CN) is required for egg activation. Moreover, we demonstrate that parthenogenetic activation of metaphase I-arrested eggs by MEK inhibition, independent of a Ca 2+ increase, requires the activity of a second egg phosphatase: PP2A. Furthermore, PP2A activity, together with CN, is required for normal egg activation during fertilisation. As ascidians are a sister group of the vertebrates, we discuss these findings in relation to cell cycle arrest and egg activation in chordates. KEY WORDS: CaMKII, PP2A, Calcineurin, Meiotic arrest, Egg activation INTRODUCTIONUnfertilised animal eggs are, in general, cell cycle arrested to prevent parthenogenetic activation by a cytoplasmic activity called cytostatic factor (CSF) first identified in 1971 in amphibian eggs (Masui and Markert, 1971) and it is the fertilising sperm that induces resumption of the cell cycle. Two aspects have been extremely well conserved during animal evolution: (1) sperm activate eggs by triggering an increase in the intracellular Ca 2+ ion concentration ( [Ca 2+ i ]) in virtually all metazoan eggs that have been studied (Stricker, 1999); and (2) cell cycle arrest of mature eggs from species ranging from cnidarians to mammals is brought about by (Dumollard et al., 2011), metaphase II arrest in vertebrates, and G1 arrest in starfish and jellyfish eggs (Kishimoto, 2003;Amiel et al., 2009). In all these cases, egg activation can be induced by the sperm-triggered Ca 2+ signal or by lowering the activity of the Mos/MEK/MAPK pathway (reviewed by Whitaker, 2006). Despite what appear to be a universal set of molecular mechanisms, it is not known how the sperm-triggered increase in [Ca 2+ i ] activates eggs arrested at metaphase I. RESEARCH ARTICLEMetaphase II-CSF in vertebrates (reviewed by Masui, 2000;Kishimoto, 2003) is now known to involve the Mos/MEK/MAPK kinase cascade and activity of the Cdc20-activated anaphase promoting complex/cyclosome (APC/C cdc20 ) inhibitor Emi2, also known as Erp1 in Xenopus Tung et al., 2005;Shoji e...

Signaling Enzymes Required for Sperm Maturation and Fertilization in Mammals

“…More than two decades ago a role for a calcium regulated protein phosphatase was proposed in the regulation of sperm motility (Tash et al, 1988;Tash and Bracho, 1994). In non-mammalian sperm, calcineurin has been shown to have role in activation of progressive motility and egg activation (Levasseur et al, 2013;Krapf et al, 2014). The catalytic and regulatory subunits of calcineurin are present as testis-specific isoforms, PPP3CC and PPP3R2.…”

α-endosulfine (ENSA) regulates exit from prophase I arrest in mouse oocytes

“…Such differences between species are not unprecedented, with one example in female meiosis being the phosphatase calcineurin. Calcineurin is important for exit from metaphase II arrest in Xenopus, exit from metaphase I arrest in an ascidian species, and progression of Drosophila oocytes through meiosis, [48][49][50] whereas there is no evidence for calcineurin functioning in exit from metaphase II arrest in mouse. 51 The defect in meiotic resumption with ENSA deficiency in mouse oocytes, analogous to a G 2 -to-M transition in mitotic cells, differs from what is observed in mouse embryonic fibroblasts (MEFs) depleted of MASTL through a conditional knockout approach.…”

Postovulatory cell death: why eggs die via apoptosis in biological species with external fertilization

“…Of note, the involvement of CaMKII and calcineurin in egg activation is not evolutionary conserved. Mature eggs of many invertebrate species do not encode the CaMKII target protein Erp1 in their genomes and presumably use the “phosphatase-only” mechanism [ 54 ]. In mammalian eggs, however, exit from meiotic metaphase arrest and cell cycle resumption was found to rely solely on CaMKII activity [ 55 , 56 ].…”