Ca2+ oscillations at fertilization in mammals are regulated by the formation of pronuclei

Abstract

In mammals, the sperm triggers a series of cytosolic Ca2+oscillations that continue for ∼4 hours, stopping close to the time of pronucleus formation. Ca2+ transients are also seen in fertilized embryos during the first mitotic division. The mechanism that controls this pattern of sperm-induced Ca2+ signalling is not known. Previous studies suggest two possible mechanisms: first, regulation of Ca2+oscillations by M-phase kinases; and second, regulation by the presence or absence of an intact nucleus. We describe experiments in mouse oocytes that differentiate between these mechanisms. We find that Ca2+oscillations continue after Cdk1-cyclin B1 activity falls at the time of polar body extrusion and after MAP kinase has been inhibited with UO126. This suggests that M-phase kinases are not necessary for continued Ca2+oscillations. A role for pronucleus formation in regulating Ca2+signalling is demonstrated in experiments where pronucleus formation is inhibited by microinjection of a lectin, WGA, without affecting the normal inactivation of the M-phase kinases. In oocytes with no pronuclei but with low M-phase kinase activity, sperm-induced Ca2+ oscillations persist for nearly 10 hours. Furthermore, a dominant negative importin β that inhibits nuclear transport, also prevents pronucleus formation and causes Ca2+ oscillations that continue for nearly 12 hours. During mitosis, fluorescent tracers that mark nuclear envelope breakdown and the subsequent reformation of nuclei in the newly formed two-cell embryo establish that Ca2+ oscillations are generated only in the absence of a patent nuclear membrane. We conclude by suggesting a model where nuclear sequestration and release of a Ca2+-releasing activity contributes to the temporal organization of Ca2+ transients in meiosis and mitosis in mice.

Ca2+ Signaling During Mammalian Fertilization: Requirements, Players, and Adaptations

“…Interestingly, in Xenopus, IP 3 R1s that are more sensitive move to the cortex from the subcortex as oocytes progress to the MII stage (Boulware and Marchant 2005). Importantly, in the mouse, ER (FitzHarris et al 2003) and possibly IP 3 R1 (our unpublished data) cortical clusters disappear ahead of the termination of the oscillations, suggesting that they are not required for the persistence of oscillations. Nevertheless, the precise distribution of IP 3 R1 in eggs suggest an important role during fertilization, which may correspond to the need for localized high amplitude [Ca 2þ ] i increases to facilitate CG release to prevent polyspermy (McAvey et al 2002).…”

“…Before the initiation of maturation, the ER in mouse oocytes shows a homogeneous distribution with slight accumulation around the GV, although by the MII stage, the ER displays a fine tubular network appearance with dense accumulation in the cortex (Mehlmann et al 1995), which is thought to facilitate the initiation of sperm-induced [Ca 2þ ] i oscillations (Kline et al 1999). The dramatic reorganization ensues at about the time of GVBD and is underpinned by distinct components of the cytoskeleton (FitzHarris et al 2007), as the migration of the ER toward the condensing chromosomes is dependent on microtubules, whereas its dispersal from the MI spindle to the egg's cortex relies on actin microfilaments (FitzHarris et al 2003). The IP 3 R1 are also organized in cortical clusters at the MII stage (Mehlmann et al 1996;Fissore et al 1999a;Ito et al 2008a), although it remains to be established whether the same cytoskeletal mechanisms that control ER organization control IP 3 R1 distribution.…”

“…IP 3 R1 phosphorylation within Cdk1 and MAPK consensus sites was reported in Xenopus oocytes and independent activation of these kinases increased IP 3 R-mediated Ca 2þ release ). Notably, an earlier study in mouse zygotes had dismissed the role of MPF and MAPK on [Ca 2þ ] i oscillations, as the continuation was unaltered by the decline in MPF activity, which occurs at the time of second PB extrusion, or after inhibition of the MAPK pathway with U0126 (Marangos et al 2003). Importantly, the phosphorylation status of IP 3 R1 was not examined in that study, and subsequent results found that IP 3 R1 MPM-2 phosphorylation outlasts MPF activity (Lee et al 2006) and that 4 hours U0126 exposure does not eliminate MPM-2 IP 3 R1 reactivity (our unpublished observations).…”

Regulation of intracellular pH during oocyte growth and maturation in mammals

“…One plausible possibility relates to the repetitive pulsatile increases in [Ca 2C ] i , which occur after fertilization to trigger development. The re-activation of pH i regulation (when MAPK activity decreases) corresponds well with the cessation of Ca 2C oscillations (Jones et al 1995, Marangos et al 2003. Some pH-regulatory transporters are sensitive to Ca 2C or its downstream pathways, and thus inactivation of pH i -regulatory transporters may prevent Ca 2C -triggered pH i fluctuations following fertilization.…”

Egg activation is the result of calcium signal summation in the mouse

“…They begin a few minutes after gamete fusion (Cuthbertson 1983, Lawrence et al 1997, Deguchi et al 2000, occur at various frequencies and cease at the time of pronucleus (PN) formation, i.e. 4 -6 h later (Jellerette et al 2000, Marangos et al 2003. However, despite increasing knowledge about signal transduction mechanisms, the functional significance of these signalling events and the rules according to which Ca 2þ signals are processed, such as frequency encoding or spike counting (Berridge 1997), or simply the summation of the biochemical effects caused by a train of [Ca 2þ ] cyt transients, are not very clear.…”