Fertilization promotes a calcium surge necessary to ensure the success of embryonic development. It seems that calcium activates apparently opposite molecular signalling pathways to achieve that end.
Sexual reproduction relies on two cellular processes: meiosis, through which two cellular divisions produce gametes (sperm and egg), and fertilization, whereby male and female gametes fuse to form a zygote. In most organisms, the egg must halt meiosis to prevent embryonic development in the absence of fertilization. So a vertebrate egg remains arrested at the metaphase stage of the second meiotic division for many hours, awaiting the sperm. If sperm arrives, this breaks the arrest by increasing the intracellular concentration of calcium ions (Ca2+), allowing cell divisions that ultimately produce a multicellular organism. Two reports1,2 published in this issue show that the enzyme calcineurin has a previously unidentified function that is triggered by the sperm-induced Ca2+ surge and is essential for overcoming the meiotic arrest.
Entry of cells into the division phase of the cell cycle (M phase) is mediated by a protein known as M-phase-promoting factor (MPF)3, which consists of a catalytic subunit, Cdk1, and a regulatory subunit, cyclin B. To overcome meiotic arrest, eggs must destroy cyclin B, thereby inactivating MPF. In vertebrate eggs, an unidentified agent dubbed cytostatic factor (CSF) mediates meiotic arrest3 by inhibiting a protein complex known as APC/C, which would otherwise destroy cyclin B.
Meiotic arrest mediated by CSF is ensured through the activity of the Mos enzyme, which is present only during meiosis, and its downstream targets, MEK, MAPK and p90Rsk (ref. 4). The relationship between the Mos–MEK–MAPK–p90Rsk pathway and APC/C was clarified5 by the discovery of an egg-specific APC/C inhibitor, Erp1 (also known as Emi2), which, when phosphorylated by p90Rsk, leads to meiotic arrest6,7. This suggests that CSF consists of Mos, its downstream enzymes and their effector, Erp1, inhibiting APC/C to stabilize MPF (Fig. 1, overleaf).
So where does calcium come into the picture? For more than a decade, the only known function of Ca2+ waves in meiotic exit was in activation of the enzyme Ca2+/calmodulin-dependent protein kinase II (CaMKII)8. Kinases regulate the activity of other proteins by phosphorylating them. CaMKII was shown9,10 to add an additional phosphate group to Erp1, allowing its subsequent phosphorylation by the Plx1 enzyme, which targets Erp1 for degradation. Free of Erp1, APC/C induces cyclin-B degradation, inactivating the kinase activity of MPF and thereby breaking meiotic arrest.
Nishiyama et al.1 (page 341) and Mochida and Hunt2 (page 336) now show that another enzyme is also essential for overcoming meiotic arrest. This is a Ca2+-dependent phosphatase known as calcineurin. Phosphatases regulate other proteins by dephosphorylating them. Working with frog eggs, the authors show that, after Ca2+ release, calcineurin is quickly and transiently activated, independently of CaMKII. Moreover, calcineurin inhibition impairs several events that normally occur in response to Ca2+ (Fig. 2). These include cyclin-B degradation, dephosphorylation of proteins phosphorylated during M phase, migration of pronuclei (gamete nuclei seen after sperm enters the egg but before the two nuclei fuse) and rearrangement of the cytoskeleton in the cortical areas of the cell — that is, areas immediately beneath the cell membrane.
Both groups1,2 therefore conclude that, in addition to the CaMKII spike, activation of calcineurin is required to break meiotic arrest imposed by the Mos–MEK–MAPK–p90Rsk–Erp1 signalling pathway, as well as for the completion of other cytoplasmic events that characterize fertilization (Fig. 2). Also noteworthy are observations1 that indicate that, to allow both the growth of sperm aster — the array of sperm microtubules that mediates chromosome separation — and the migration of male and female pronuclei towards each other to fuse and form the nucleus of the zygote, calcineurin activation must be transient.
In contrast to CaMKII, the molecular targets of which are well defined, proteins dephosphorylated by calcineurin to break CSF-mediated meiotic arrest have not been identified. Calcineurin could promote CaMKII-independent cyclin-B degradation by regulating Erp1 or APC/C, or both. As meiotic arrest by Erp1 depends on its phosphorylation by p90Rsk (refs 6, 7), calcineurin could also dephosphorylate p90Rsk-phosphorylated sites on Erp1, allowing its inactivation before it is degraded by the CaMKII-mediated pathway.
Other substrate candidates for calcineurin include APC/C components. Mochida and Hunt2 show that, after the addition of Ca2+ to egg extracts, calcineurin quickly dephosphorylates an APC/C component, Apc3, as well as the APC/C activator Cdc20. Whether, in addition to Erp1 degradation, activation of APC/C depends on dephosphorylation of its subunits by calcineurin remains to be determined.
It is not unlikely that calcineurin also directly affects MPF. Mochida and Hunt show that this phosphatase promotes cyclin-B dephosphorylation, which could rapidly inactivate MPF before Erp1 is degraded. One could also imagine that, by directly or indirectly controlling Cdk1 phosphorylation, calcineurin would inhibit the catalytic activity of MPF without affecting cyclin B. Thus, simultaneous and parallel activities of a phosphatase (calcineurin) and a kinase (CaMKII) — both activated by Ca2+ and converging to inactivate MPF — would ensure rapid and irrevocable exit from meiosis.
Mochida and Hunt2 find that calcineurin is also essential for reversing the phosphorylation state of other proteins specific to the second meiotic division. They add another twist to the story, showing that, shortly after the calcineurin spike, a second (non-calcineurin) wave of phosphatase activity fluctuates in a cell-cycle-dependent manner, presumably to dephosphorylate proteins common to meiotic and mitotic M phase. So whereas calcineurin and/or CaMKII might not be required for mitotic exit, the rapid embryonic cell cycles that follow fertilization must be controlled by other mitotic kinases and phosphatases.
Because in vertebrates both CaMKII and calcineurin are devoted to breaking the meiotic arrest at metaphase, these enzymes must have fertilization-specific substrates. These could include protein(s) that suppress a premature second wave of phosphatase activity before calcineurin activity peaks, and probably cytoskeletal proteins and their regulators. Fertilization triggers dramatic changes in the cytoskeleton and induces migration of pronuclei, which, as Nishiyama and colleagues1 find, are also under calcineurin control (Fig. 2).
Depending on the species, eggs await fertilization either in the M-phase or the interphase stage of the cell cycle. Therefore, molecular pathways ensuring meiotic arrest could be active both in the presence and in the absence of MPF activity. In both cases, however, the Mos–MEK–MAPK–p90Rsk pathway and fertilization-associated Ca2+ surge remain essential for releasing meiotic arrest. Among the questions that the work of Nishiyama et al.1 and Mochida and Hunt2 raises, is whether, although CaMKII and Erp1 seem to be specific to eggs arrested in the M phase, calcineurin is universal in initiating embryonic development throughout the animal kingdom.
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Conformational transition pathway in the allosteric process of calcium-induced recoverin: Molecular dynamics simulations
Journal of Computational Chemistry (2009)